Cell culture medium

ABSTRACT

The present disclosure related to isolated laminin-521, methods for making recombinant laminin-521, host cells that express recombinant laminin-521, and compositions containing laminin-521. Laminin-521 can maintain stem cells in vitro pluripotency, enable self-renewal, and enable single cell survival of human embryonic stem cells. When pluripotent human embryonic stem cells are cultured on plates coated with recombinant laminin-521 (laminin-11), in the absence of differentiation inhibitors or feeder cells, the embryonic stem cells proliferate and maintain their pluripotency. It has also been discovered that human recombinant laminin-521 (laminin-11) provides single cell survival of stem cells after complete dissociation into a single cell suspension. Useful cell culture mediums containing at most 3.9 ng/ml of beta fibroblast growth factor (bFGF) are also described herein.

This application claims priority from U.S. Provisional PatentApplication Ser. No. 61/424,273, filed Dec. 17, 2010; U.S. ProvisionalPatent Application Ser. No. 61/488,353, filed May 20, 2011; U.S.Provisional Patent Application Ser. No. 61/533,063, filed Sep. 9, 2011;U.S. Provisional Patent Application Ser. No. 61/537,940, filed Sep. 22,2011; U.S. Provisional Patent Application Ser. No. 61/565,380, filedNov. 30, 2011; and U.S. Provisional Patent Application Ser. No.61/565,849, filed Dec. 1, 2011. The entireties of those provisionalapplications are fully incorporated by reference herein.

BACKGROUND

This application relates to cell biology, cell differentiation, celltherapy, molecular biology, proteins, recombinant human proteins,nucleic acids, and laminins.

Basal laminae (basement membranes) are sheet-like, cell-associatedextracellular matrices that play a central role in cell growth, cellulardifferentiation, cell phenotype maintenance, tissue development, andtissue maintenance. They are present in virtually all tissues, andappear in the earliest stages of embryonic development.

Basal laminae are central to a variety of architectural andcell-interactive functions. For example:

1. They serve as architectural supports for tissues, providing adhesivesubstrata for cells.

2. They create perm-selective barriers between tissue compartments thatimpede the migration of cells and passively regulate the exchange ofmacromolecules. These properties are illustrated by the kidneyglomerular basement membrane, which functions as an important filtrationstructure, creating an effective blood-tissue barrier that is notpermeable to most proteins and cells.

3. Basal laminae create highly interactive surfaces that can promotecell migration and cell elongation during embryogenesis and woundrepair. Following an injury, they provide a surface upon which cellsregenerate to restore normal tissue function.

4. Basal laminae present information encoded in their structure tocontacting cells that is important for cellular differentiation,prevention of apoptosis, and tissue maintenance. This information iscommunicated to the cells through various receptors that include theintegrins, dystroglycan, and cell surface proteoglycans. Signaling isdependent not only on the presence of matrix ligands and correspondingreceptors that interact with sufficient affinities, but also on suchtopographical factors as ligand density in a three-dimensional matrix“landscape”, and on the ability of basal lamina components to clusterreceptors. Because these matrix proteins can be long-lived, basallaminae create a “surface memory” in the basal lamina for resident andtransient cells.

The basal lamina is largely composed of laminin and type IV collagenheterotrimers that in turn become organized into complex polymericstructures. Additional components include proteoglycans such as agrinand perlecan and nidogens (entactins). To date, six type IV collagenpolypeptide chains and at least twelve laminin subunit chains have beenidentified. These chains possess shared and unique functions and areexpressed with specific temporal (developmental) and spatial(tissue-site specific) patterns.

Laminins are a family of heterotrimeric glycoproteins that resideprimarily in the basal lamina. They function via binding interactionswith neighboring cell receptors on the one side, and by binding to otherlaminin molecules or other matrix proteins such as collagens, nidogensor proteoglycans. The laminin molecules are also important signalingmolecules that can strongly influence cellular behavior and function.Laminins are important in both maintaining cell/tissue phenotype, aswell as in promoting cell growth and differentiation in tissue repairand development.

Laminins are large, multi-domain proteins, with a common structuralorganization. The laminin molecule integrates various matrix and cellinteractive functions into one molecule.

A laminin protein molecule comprises one α-chain subunit, one β-chainsubunit, and one γ-chain subunit, all joined together in a trimerthrough a coiled-coil domain. FIG. 1 depicts the resulting structure ofthe laminin molecule. The twelve known laminin subunit chains can format least 15 trimeric laminin types in native tissues. Within thetrimeric laminin structures are identifiable domains that possessbinding activity towards other laminin and basal lamina molecules, andmembrane-bound receptors. FIG. 2 shows the three laminin chain subunitsseparately. For example, domains VI, IVb, and IVa form globularstructures, and domains V, IIIb, and IIIa (which contain cysteine-richEGF-like elements) form rod-like structures. Domains I and II of thethree chains participate in the formation of a triple-strandedcoiled-coil structure (the long arm).

There exist five different alpha chains, three beta chains and threegamma chains that in human tissues have been found in at least fifteendifferent combinations. These molecules are termed laminin-1 tolaminin-15 based on their historical discovery, but an alternativenomenclature describes the isoforms based on their chain composition,e.g. laminin-111 (laminin-1) that contains alpha-1, beta-1 and gamma-1chains. Four structurally defined family groups of laminins have beenidentified. The first group of five identified laminin molecules allshare the β1 and γ1 chains, and vary by their α-chain composition (α1 toα5 chain). The second group of five identified laminin molecules,including laminin-521, all share the β2 and γ1 chain, and again vary bytheir α-chain composition. The third group of identified lamininmolecules has one identified member, laminin-332, with a chaincomposition of α3β3β2. The fourth group of identified laminin moleculeshas one identified member, laminin-213, with the newly identified γ3chain (α2β1γ3).

There have been no reports of isolated laminin-521 that is free of otherlaminin chains. Thus far, there are no studies on the function oflaminin-521. Attempts to purify laminin-521 from cell sources byaffinity chromatography using laminin chain antibodies have beenunsuccessful in eliminating, for example, laminin β1 chain, which is acomponent of laminin-411 and laminin-511. It would be desirable toprovide compositions that contain laminin-521 (aka LN-521) and methodsfor making laminin-521.

Human embryonic stem (hES) cells hold promise for the development ofregenerative medicine for a variety of diseases, such as spinal cord andcardiac injuries, type I diabetes and neurodegenerative disorders likeParkinson's disease. A stem cell is an undifferentiated cell from whichspecialized cells are subsequently derived. Embryonic stem cells possessextensive self-renewal capacity and pluripotency with the potential todifferentiate into cells of all three germ layers. They are useful fortherapeutic purposes and may provide unlimited sources of cells fortissue replacement therapies, drug screening, functional genomics andproteomics.

A prerequisite for the development of stem cell derived cells forregenerative medicine are methods that allow long-term cultures ofpluripotent stem cells, chemically defined and repeatabledifferentiation protocols, as well as xeno-free cell culture systems.However, culturing of pluripotent human embryonic stem cells (hES cells)and induced pluripotent stem cells (hiPS cells) has encountered a numberof problems. One major problem has been that hES cells grow slowly inclusters that need to be manually split for cell propagation.Dissociation of the cells usually leads to extensive cell death, thecloning efficiency of hES cells after complete dissociation being 5-1%.

Maintenance of pluripotent hES cells has required complex culturesubstrata, such as extracellular matrix protein mixtures like the mousetumor derived Matrigel or fibroblast feeder cell layers, that may beimmunogenic and toxic and that generate extensive batch to batchvariability reducing reliability of the experiments. Thus far, the mostsuccessful feeder cell free substrate used for hES cell cultures isMatrigel, a complex tumor and BM-like extract obtained from murineEngelbreth-Holm-Swarm (EHS) sarcoma tumor tissues. Matrigel mainlycontains murine LN-111, type IV collagen, perlecan and nidogen, but alsovarying amounts of other materials, including growth factors andcellular proteins and, therefore, its composition is undefined andvaries from batch-to-batch. This variability can cause irreproducibilityof scientific results, and due to the animal origin of the substratummakes Matrigel unacceptable for the expansion and maintenance of hEScells for human cell therapy.

However, successful development of more or less defined coatingmaterials that support self-renewal of hES and hiPS cells has recentlybeen reported. It has been reported that recombinant vitronectinsupports adhesion and self-renewal of hES cells. An acrylate coatingcontaining a variety of peptides from various ECM proteins has also beenpreviously developed, and it has been shown that a syntheticmethacrylate-based polymer also facilitated adhesion and self-renewal ofhES cells.

One of the main problems with large-scale propagation of hES cells isthat they poorly survive replating after dissociation into single cellsuspension. This, in turn, makes passaging tedious and large-scaleautomated expansions impossible. However, hES cells released into singlecell suspension using trypsin treatment in the presence of a rho-kinase(ROCK) inhibitor¹⁰ or blebbistatin¹¹ can be plated and expanded fromsingle clones, but the molecules are not components of the natural stemcell niche, and they affect the actin cytoskeleton and thus can causecell damage. Therefore, the use of the ROCK inhibitor may not be apreferred solution for long-term expansion of hES cells aimed for celltherapy purposes.

For the purposes of regenerative medicine, there is a desire to developmethods that allow derivation and long-term cultures of pluripotent stemcells under chemically defined, xeno-free, pathogen-free, and stablebatch-to-batch conditions. Moreover, such methods should allow fast andeconomically efficient scale-up to acquire large quantities ofpluripotent hES/hiPS cells in a short period of time. Preferably, themethods should also allow clonal survival of human ES cells in mediacontaining no synthetic inhibitor of apoptosis, that could facilitatescientific and clinical applications involving cell sorting or geneknock-out in the cells.

BRIEF DESCRIPTION

The present disclosure provides isolated laminin-521 (also known asLN-521 or laminin-11) and methods for producing isolated laminin-521. Infurther aspects, the present disclosure provides recombinant host cellsthat express laminin-521 chains and secrete recombinant laminin-521.

In other aspects, the present disclosure provides GMP qualitylaminin-521 for culturing cells for differentiation and maintenance forthe purpose of developing cells for human cell therapy. The presentdisclosure also provides pharmaceutical compositions, comprisingisolated laminin-521 together with a pharmaceutically acceptablecarrier. Such pharmaceutical compositions can optionally be providedwith other extracellular matrix components.

The present disclosure also provides methods to effectively generateamounts of isolated laminin-521 for various uses. In preferredembodiments of those uses, recombinant laminin-521 is used. Kitscomprising an amount of isolated laminin-521, or pharmaceuticalcompositions thereof, effective for the desired effect, and instructionsfor the use thereof, are also disclosed.

The present disclosure also provides methods for culturing stem cells inmonolayer cultures which facilitates cellular homogeneity, removal ofthe stem cells from a cell culture plate or other cellular support insingle cell suspension, and replating stem cells in single cellsuspension for passaging and expansion in significant dilutions thatenable expansion of stem cell cultures and large scale production ofsuch cells.

In further aspects, the present disclosure describes culturingpluripotent stem cells on laminin-521 in monolayer culture, removingstem cells from the cell culture plates along with other cell supportingmaterials in single cell suspension, and replating the stem cells fromsingle cell suspension as single cells on a matrix containinglaminin-521 such that this process can be performed in automated roboticsystems with high efficiency.

In further aspects, the present disclosure provides improved medicaldevices and grafts, wherein the improvement comprises providing medicaldevices and grafts with an effective amount of isolated laminin-521.

In further aspects, the disclosure provides improved cell culturedevices, and methods for preparing improved cell culture devices, forthe growth and maintenance of phenotypes of cells in culture, byproviding an effective amount of isolated laminin-521 to a cell culturedevice for cell attachment, and subsequent cell stasis, proliferation,differentiation, and/or migration.

In other aspect, the disclosure provides compositions, such as humanrecombinant laminin-521, and methods for culturing and rapid expandingof human embryonic stem cells in vitro in undifferentiated state. Themethods comprise single cell dissociation of human embryonic stem cellsand plating them on laminin-521 coated cell culture dishes in mediumwithout any Rho-associated kinase (ROCK) inhibitor (e.g. withoutY-27632) (Watanabe et al., Nature Biotechnology 25, 681-686 (2007)). Theimprovement provides a possibility to expand human embryonic stem cellsin pluripotent state faster than in conventional human embryonic stemcell cultures.

In further aspects, the disclosure provides new materials, such as humanrecombinant laminin-521, which permit human embryonic cell survivalafter dissociation into single cell suspension. The improvement providesbetter cloning survival of human embryonic stem cells, which may beadvantageous for isolating clones (e.g. after genetic manipulations) orobtaining homogeneous and uniformal human embryonic stem cellpopulations.

Disclosed in some embodiments is isolated recombinant laminin-521,comprising: an alpha chain comprising a polypeptide with at least 80%identity to a polypeptide sequence of SEQ ID NO: 1; a beta chaincomprising a polypeptide with at least 70% identity to a polypeptidesequence of SEQ ID NO: 2; and a gamma chain comprising a polypeptidewith at least 70% identity to a polypeptide sequence of SEQ ID NO: 3;wherein the alpha, beta, and gamma chains are assembled into recombinantlaminin-521.

In further embodiments, the alpha chain polypeptide has at least 90%identity to the polypeptide sequence of SEQ ID NO: 1. The beta chainpolypeptide may also have at least 90% identity to the polypeptidesequence of SEQ ID NO: 2. The gamma chain polypeptide may also have atleast 90% identity to the polypeptide sequence of SEQ ID NO: 3.

In other embodiments, the alpha chain has the polypeptide sequence ofSEQ ID NO: 1. Furthermore, the beta chain may have the polypeptidesequence of SEQ ID NO: 2. Even more specifically, the gamma chain mayhave the polypeptide sequence of SEQ ID NO: 3.

The beta chain polypeptide may have at least 80% identity to thepolypeptide sequence of SEQ ID NO: 2. The gamma chain polypeptide mayhave at least 80% identity to the polypeptide sequence of SEQ ID NO: 3.

Also disclosed in embodiments is a pharmaceutical composition,comprising: a) the isolated recombinant laminin-521 of claim 1; and b) apharmaceutically acceptable carrier.

Also disclosed in embodiments is a composition that enables self-renewalof pluripotent stem cells grown in vitro, comprising a growth medium anda coating thereover, the coating comprising recombinant laminin 521(laminin-11).

The composition may further comprise a growth factor.

The composition may be devoid of any differentiation inhibitors. Thecomposition may also be devoid of any feeder cells. The composition mayalso be devoid of any differentiation inductors. In some embodiments,the composition is devoid of any differentiation inhibitors, feedercells, or differentiation inductors.

Also disclosed in embodiments is a method for maintaining thepluripotency of pluripotent stem cells in vitro, comprising: providing asubstrate comprising a growth medium and a coating thereover, thecoating comprising recombinant laminin 521 (laminin-11); dissociatingpluripotent stem cells into a single cell suspension; and placing thepluripotent stem cells in the single cell suspension on the coating.

The growth medium may comprise a growth factor or growth factors.Exemplary growth factors include basic fibroblast growth factor andinsulin growth factor.

Sometimes, the growth medium and the coating are devoid of anydifferentiation inhibitors. For example, leukemia inhibitor factor isnot present.

The composition may be devoid of any feeder cells, such as mousefibroblasts or human foreskin fibroblasts. The composition may be devoidof any differentiation inductors, such as Noggin or keratinocyte growthfactor. In some embodiments, the composition is devoid of anydifferentiation inhibitors, feeder cells, or differentiation inductors.

The pluripotent stem cells may be placed on the laminin-521 coating as amonolayer.

The pluripotent stem cells may be placed on the coating at a density of200 cells/mm² or less. Alternatively, the pluripotent stem cells may beplaced on the coating at a density of 200 cells/mm² or more. Thepluripotent stem cells can alternatively be placed on the coating suchthat no two stem cells contact each other.

Also disclosed in embodiments is isolated recombinant laminin-521produced by a method comprising: providing host cells that expressrecombinant laminin-521, wherein the recombinant laminin-521 comprises:a first chain comprising a polypeptide with at least 80% identity to apolypeptide sequence of SEQ ID NO: 1, a second chain comprising apolypeptide with at least 70% identity to a polypeptide sequence of SEQID NO: 2, and a third chain comprising a polypeptide with at least 70%identity to a polypeptide sequence of SEQ ID NO: 3, wherein the first,second, and third chains are assembled into recombinant laminin-521;growing the host cells in a cell culture medium under conditions tostimulate expression of the recombinant laminin-521 chains; passing thehost cell culture medium through a column, wherein the column contains acompound that binds to the recombinant laminin-521; washing the columnto remove unbound materials; and eluting the bound recombinantlaminin-521 from the column.

Described in other embodiments are methods of maintaining thepluripotency of pluripotent stem cells in vitro, comprising: receiving asubstrate having a coating thereon, the coating containing an intactlaminin; placing pluripotent stem cells and a cell culture medium on thesubstrate; and activating the PI3-kinase/Akt pathway.

The intact laminin may be laminin-521 or laminin-511. In someembodiments, the cell culture medium does not contain any growthfactors, such as beta fibroblast growth factor (bFGF). The coating mayalso contain a cadherin. The pluripotent stem cells may be placed on thecoating at a density of 200 cells/mm2 or less.

The present disclosure also relates to cell culture media that can beused to maintain stem cells in a pluripotent state. Described in variousembodiments is a cell culture medium that provides nutrition topluripotent stem cells, comprising from greater than zero to 3.9 ng/mLof basic fibroblast growth factor (bFGF).

The cell culture medium may comprise 3.5 ng/mL or less of bFGF,including from 0.5 to 3.5 ng/mL of bFGF.

The cell culture medium may further comprise at least one inorganicsalt, at least one trace mineral, at least one energy substrate, atleast one lipid, at least one amino acid, at least one vitamin, or atleast one additional growth factor. In particular embodiments, the cellculture medium further comprises at least one inorganic salt, at leastone trace mineral, at least one energy substrate, at least one lipid, atleast one amino acid, and at least one vitamin.

The cell culture medium may not contain any one of (1) albumin, (2)insulin or an insulin substitute, or (3) transferrin or a transferrinsubstitute.

The cell culture medium may further comprise albumin, insulin, lithiumchloride, GABA, TGF beta 1, pipecolic acid, L-glutamine, MEMnon-essential amino acid solution, and DMEM/F12 solution.

The cell culture medium may further comprise at least one additionalgrowth factor, at least one trace mineral, and at least one lipid.

Also disclosed is a system for maintaining pluripotent stem cells,comprising: a cell culture medium comprising from greater than zero to3.9 ng/mL of basic fibroblast growth factor (bFGF); and a substrate forproviding support to the stem cells.

The substrate may contain laminin-521 or laminin-511. The substrate mayalso contain a cadherin.

These and other non-limiting characteristics of the disclosure are moreparticularly disclosed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following is a brief description of the drawings, which arepresented for the purposes of illustrating the exemplary embodimentsdisclosed herein and not for the purposes of limiting the same.

FIG. 1 is a rotary shadowing electron microscopy picture of arecombinant laminin molecule.

FIG. 2 shows the structural motifs of laminin α, β, and γ chains. TheN-terminal, internal, and C-terminal globular domains are depicted aswhite ovals. The coiled-coil forming domains (I and II) are shown aswhite rectangles. The rod-like structures (domains V, IIIb, and IIIa)are depicted as grey rectangles.

FIG. 3 shows the results of characterization of human recombinantlaminin-521 using SDS-PAGE. An immunoblot of recombinant laminin-521 wasmade under non-reducing (labeled as ox) and reducing (labeled as red)conditions. Proteins on 3-8% gels were transferred onto PVDF membranesfollowed by staining with antibodies to laminin α5 (2F7), β2 (MAB2066),γ1 (MAB1921) and γ1 (H-19).

FIGS. 4 and 5 show the Crystal Violet staining of human ES cellsadherent to dishes coated in Matrigel (Mg), mouse laminin-111 (mLN111),human recombinant-laminin-511 (r-laminin-511 or r-LN-511), humanrecombinant-laminin-521 (r-laminin-521 or r-LN-521), or a mixture ofr-laminin-511 and r-laminin-521 (mix) after one day in culture. BothFigures are magnified 5×. In FIG. 4, the cells were cultured withoutROCK inhibitor. In FIG. 5, the cells were cultured with ROCK inhibitorY-27632.

FIG. 6 is a graph showing the adhesion of human ES cells to humanr-laminin-521 (labeled as LN521), human r-laminin-511 (LN511), andMatrigel (Mg) coated dishes after one hour in culture without ROCKinhibitor. Error bars show the standard error of measurement. (n=3).

FIG. 7 is a graph showing the adhesion of human ES cells to dishescoated in either Matrigel (Mg), mouse laminin-111 (mLN111), humanr-laminin-511 (LN511), human r-laminin-521 (LN521), or a mixture ofr-laminin-511 and r-laminin-521 (mix), after one day in culture andwithout ROCK inhibitor. Also included are results for dishes with ROCKinhibitor and either Matrigel (In&Mg), mouse laminin-111 (In&mLN111),human r-laminin-511 (In&LN511), human r-laminin-521 (In&LN521), or amixture of r-laminin-511 and r-laminin-521 (In&mix), also after one dayin culture. The cells for both experiments were plated at the samedensity on the same cell culture dish. Error bars show standard error ofmeasurement (n=3).

FIG. 8 shows the growth curves for human ES cells cultured onr-laminin-521 (LN-521_(—)1 and LN-521_(—)2) using single celldissociation passaging and for human ES cells cultured on Matrigel(Mg_(—)1 and Mg_(—)2) using passaging in small clumps. The latter cellswere passaged as described in Rodin et al., Nature Biotechnol., vol. 28,pp. 611-615 (2010).

FIG. 9 is a FACS analysis of human ES cells after several single celldissociation passages on r-laminin-521 for OCT4, a marker ofpluripotency. The percentage of positive cells is listed in parentheses.

FIG. 10 is a FACS analysis of human ES cells after several passages onMatrigel using splitting in small clumps for OCT4. The percentage ofpositive cells is listed in parentheses.

FIG. 11 shows the results of a real-time quantitative RT-PCR analysiswhich was used to compare numbers of mRNA transcripts of thepluripotency markers Oct4 and Nanog in human ES cells cultured on humanr-laminin-521 (LN521) after several single cell dissociation passagingand in the cells cultured on Matrigel (Mg) after several passaging ofthe cells in clumps. Error bars show 95% confidence intervals.

FIG. 12 is a Western blot comparing the levels of phosphorylated myosinlight chain (P-MLC) in stem cells grown on Matrigel (Mg), LN-111,LN-511, and LN-521 one hour after plating.

FIG. 13 is a Western blot comparing the levels of phosphorylated myosinlight chain (P-MLC) in stem cells grown on Matrigel (Mg), LN-111,LN-511, and LN-521 six hours after plating.

FIG. 14 is a graph comparing the relative migration of human embryonicstem cells grown on Matrigel (Mg), LN-111, LN-511, and LN-521 betweenfive and seven hours after plating.

FIG. 15 is a graph comparing the relative migration of human embryonicstem cells grown on LN-521 with IgG and IgM blocking antibodies applied(IgGM) and with a function blocking antibody to integrin β1 (b1)applied. This shows that blocking the integrin significantly reducedmotility.

FIG. 16 is a graph comparing the relative survival of hES cells onLN-521 in the presence of water, DMSO, LY294002 (Akt inhibitor),wortmannin (PI3K/Akt inhibitor), and PD 98059 (MEK1 inhibitor). Thisshowed that activation of PI3K/Akt was necessary for hES cell survivalon LN-521. The medium contained bFGF.

FIG. 17 is a graph comparing the relative survival of hES cells onLN-521 in the presence of DMSO, LY294002 (Akt inhibitor), and PD 98059(MEK1 inhibitor). No exogenous bFGF was present.

FIG. 18 is a Western blot comparing cells treated with LY 294002 againstcontrol cells (DMSO) and collected one hour after plating on LN-521.

FIG. 19 is a Western blot comparing cells treated with PD98059 againstcontrol cells (DMSO) and collected one hour after plating on LN-521.

FIG. 20 is a graph showing the relative levels of Akt2 phosphorylationon cell lysates collected one hour after plating on Matrigel (Mg),LN-111, LN-511, or LN-521, obtained using ELISA.

FIG. 21 is a graph showing the relative levels of Akt1 phosphorylationon cell lysates collected one hour after plating on Matrigel (Mg),LN-111, LN-511, or LN-521, obtained using ELISA.

FIG. 22 is a graph showing the growth curve of HS181 hES cells culturedin a low bFGF medium (O3_(—)3.9) compared to a higher bFGF medium(O3_(—)100).

FIG. 23 is a graph showing the relative mRNA expression level for twopluripotency markers (Oct4 and Nanog) in HS181 hES cells cultured in alow bFGF medium (O3_(—)3.9) compared to a higher bFGF medium(O3_(—)100).

FIG. 24 is a picture showing an early stage derivation of a new humanembryonic stem HS841 cell line on laminin-521/E-Cadherin matrix.

DETAILED DESCRIPTION

A more complete understanding of the compositions and methods disclosedherein can be obtained by reference to the accompanying drawings. Thesefigures are merely schematic representations based on convenience andthe ease of demonstrating the present disclosure, and are, therefore,not intended to define or limit the scope of the exemplary embodiments.

Although specific terms are used in the following description for thesake of clarity, these terms are intended to refer only to theparticular structure of the embodiments selected for illustration in thedrawings, and are not intended to define or limit the scope of thedisclosure. In the drawings and the following description below, it isto be understood that like numeric designations refer to components oflike function.

All publications, patents, and patent applications discussed herein arehereby incorporated by reference in their entirety.

Unless otherwise stated, the techniques utilized in this application maybe found in any of several well-known references such as: MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P.Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide toMethods and Applications (Innis, et al. 1990. Academic Press, San Diego,Calif.), Culture of Animal Cells: A Manual of Basic Technique, SecondEd. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), or the Ambion 1998 Catalog (Ambion, Austin, Tex.).

As used herein, the term “isolated nucleic acid sequence” refers to anucleic acid sequence that is free of gene sequences which naturallyflank the nucleic acid in the genomic DNA of the organism from which thenucleic acid is derived (i.e., genetic sequences that are locatedadjacent to the gene for the isolated nucleic molecule in the genomicDNA of the organism from which the nucleic acid is derived). The“isolated” sequence may, however, be linked to other nucleotidesequences that do not naturally flank the recited sequence, such as aheterologous promoter sequence, or other vector sequences. It is notnecessary for the isolated nucleic acid sequence to be free of othercellular material to be considered “isolated”, as a nucleic acidsequence according to the disclosure may be part of an expression vectorthat is used to transfect host cells (see below).

The present disclosure provides recombinant expression vectorscomprising a full length laminin β2 chain nucleic acid sequence (SEQ IDNO: 4) of the human laminin in chain. In some embodiments, theexpression vectors comprise a nucleic acid encoded by SEQ ID NO: 4,operatively linked to a heterologous promoter (i.e. is not the naturallyoccurring promoter for the given β2 laminin chain). A promoter and alaminin β2 chain nucleic acid sequence are “operatively linked” when thepromoter is capable of driving expression of the laminin β2 chain DNAinto RNA.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA into which additional DNA segments may be cloned.Another type of vector is a viral vector, wherein additional DNAsegments may be cloned into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors), are integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” or simply “expression vectors”. In the presentdisclosure, the expression of the laminin polypeptide sequence isdirected by the promoter sequences of the disclosure, by operativelylinking the promoter sequences of the disclosure to the gene to beexpressed. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably, asthe plasmid is the most commonly used form of vector. However, thedisclosure is intended to include other forms of expression vectors,such as viral vectors (e.g., replication defective retroviruses,adenoviruses and adeno-associated viruses), which serve equivalentfunctions.

The vector may also contain additional sequences, such as a polylinkerfor subcloning of additional nucleic acid sequences, or apolyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the methods of the disclosure,and any such sequence may be employed, including but not limited to theSV40 and bovine growth hormone poly-A sites. Also contemplated as anelement of the vector is a termination sequence, which can serve toenhance message levels and to minimize readthrough from the constructinto other sequences. Additionally, expression vectors typically haveselectable markers, often in the form of antibiotic resistance genes,that permit selection of cells that carry these vectors.

In further embodiments, the present disclosure provides host cellstransfected with the laminin β2 chain-expressing recombinant expressionvectors disclosed herein. As used herein, the term “host cell” isintended to refer to a cell into which a nucleic acid of the presentdisclosure, such as a recombinant expression vector, has beenintroduced. Such cells may be prokaryotic, which can be used, forexample, to rapidly produce a large amount of the expression vectors ofthe disclosure, or may be eukaryotic, for functional studies.

The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It should be understood that such terms refernot only to the particular subject cell but to the progeny or potentialprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

The host cells can be transiently or stably transfected with one or moreof the expression vectors of the disclosure. Such transfection ofexpression vectors into prokaryotic and eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2.sup.nd Ed. (R. I. Freshney. 1987. Liss,Inc. New York, N.Y.).

In another aspect, the present disclosure provides an isolated fulllength human laminin β2 chain polypeptide consisting of the amino acidsequence of SEQ ID NO: 2.

As used herein, an “isolated polypeptide” refers to a polypeptide thatis substantially free of other proteins, including other laminin chains,and gel agents, such as polyacrylamide and agarose. In preferredembodiments, the isolated laminin polypeptide is free of detectablecontaminating laminin chains. Thus, the protein can either be isolatedfrom natural sources, or recombinant protein can be isolated from thetransfected host cells disclosed above.

In another aspect, the present disclosure provides isolated laminin-521.As used herein, the term “laminin-521” refers to the protein formed byjoining α5, β2 and γ1 chains together. The term should be construed asencompassing both recombinant laminin-521 and heterotrimeric laminin-521from naturally occurring sources. In preferred embodiments, thelaminin-521 comprises recombinant laminin-521 (or “r-laminin-521”).

As used herein, the term “r-laminin-521” refers to recombinantheterotrimeric laminin-521, expressed by a host cell that has beentransfected with one or more expression vectors comprising at least onenucleic acid sequence encoding a laminin-521 chain selected from the α5,β2 and γ1 chains, or processed or secreted forms thereof. Suchr-laminin-521 can thus comprise α5, β2, and γ1 sequences from a singleorganism, or from different organisms. Various laminin-521 chain DNAsequences are known in the art, and the use of any such sequence toprepare the r-laminin-521 of the disclosure is contemplated. (See, forexample, Pouliot, N. et al., Experimental Cell Research 261(2):360-71,(2000); Kikkawa, Y. et al., Journal of Cell Science 113 (Pt 5):869-76,(2000); Church, H J. et al., Biochemical Journal 332 (Pt 2):491-8,(1998); Sorokin, L M. et al., Developmental Biology 189(2):285-300,(1997); Miner, J H. et al., Journal of Biological Chemistry270(48):28523-6, (1995); Sorokin, L. et al., European Journal ofBiochemistry 223(2):603-10, (1994)). In preferred embodiments, ther-laminin-521 is formed from recombinant human α5, β2, and γ1polypeptide chains.

The disclosure encompasses those laminin molecules wherein only one ortwo chains that make up the recombinant heterotrimeric laminin-521 areencoded by endogenous laminin-521 chains. In preferred embodiments, eachof the α5, β2, and γ1 polypeptide chains are expressed recombinantly.

The laminin-521 is an intact protein. The term “intact” refers to theprotein being composed of all of the domains of the α-chain, β-chain,and γ-chain, with the three chains being joined together to form theheterotrimeric structure. The protein is not broken down into separatechains, fragments, or functional domains. The term “chain” refers to theentirety of the alpha, beta, or gamma chain of the laminin protein. Theterm “fragment” refers to any protein fragment which contains one, two,or three functional domains that possesses binding activity to anothermolecule or receptor. However, a chain should not be considered afragment because each chain possesses more than three such domains.Similarly, an intact laminin protein should not be considered afragment. Examples of functional domains include Domains I, II, III, IV,V, VI, and the G domain.

Laminin-521 is a secreted protein, which is capable of being directed tothe endoplasmic reticulum (ER), secretory vesicles, and theextracellular space as a result of a signal sequence. If the secretedprotein is released into the extracellular space, the secreted proteincan undergo extracellular processing to produce a “mature” protein. Suchprocessing events can be variable, and thus may yield different versionsof the final “mature protein”. For example, the lengths of the α5, β2,and γ1 chains may vary between proteins. However, the final matureprotein still has the same functionality, even though the chain lengthsvary. The isolated laminin-521 of the present disclosure includesheterotrimers comprising both the full length polypeptide chains and anysuch naturally processed laminin-521 polypeptide chains.

As used herein, a laminin-521 polypeptide chain refers to a polypeptidechain according to one or more of the following:

(a) a polypeptide chain that comprises a polypeptide structure selectedfrom the group consisting of: R1-R2-R3, R1-R2-R4, R3, R4, R1-R3, R1-R4,R2-R3, and R2-R4, wherein R1 is an amino terminal methionine; R2 is asignal sequence that is capable of directing secretion of thepolypeptide, wherein the signal sequence may be the natural signalsequence for the particular laminin chain, that of another secretedprotein, or an artificial sequence; R3 is a secreted laminin chainselected from the group consisting of a α5 chain, a β2 chain, and a γ1chain; and R4 is a secreted α5, β2, or γ1 laminin chain that furthercomprises an epitope tag (such as those described below), which can beplaced at any position within the laminin chain amino acid sequence; or

(b) a polypeptide chain that is encoded by a polynucleotide thathybridizes under high or low stringency conditions to the codingregions, or portions thereof, of one or more of the recombinantlaminin-521 chain DNA sequences disclosed herein (SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 6), or complementary sequences thereof; or

(c) a polypeptide chain that has at least 70% identity to one or more ofthe disclosed laminin-521 polypeptide chain amino acid sequences (SEQ IDNO: 1, SEQ ID NO: 2, SEQ ID NO: 3), preferably at least 80% identity,and most preferably at least about 90% identity.

“Stringency of hybridization” is used herein to refer to washingconditions under which nucleic acid hybrids are stable. The disclosurealso includes nucleic acids that hybridize under high stringencyconditions (as defined herein) to all or a portion of the codingsequences of the laminin chain polynucleotides disclosed herein, ortheir complements. The hybridizing portion of the hybridizing nucleicacids is typically at least 50 nucleotides in length. As known to thoseof ordinary skill in the art, the stability of hybrids is reflected inthe melting temperature (T_(M)) of the hybrids. T_(M) decreasesapproximately 1-1.5° C. with every 1% decrease in sequence homology. Ingeneral, the stability of a hybrid is a function of sodium ionconcentration and temperature. Typically, the hybridization reaction isperformed under conditions of lower stringency, followed by washes ofvarying, but higher, stringency. As used herein, high stringency refersto an overnight incubation at 42° C. in a solution comprising 50%formamide, 5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20μg/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65° C.

Also contemplated are laminin-521-encoding nucleic acid sequences thathybridize to the polynucleotides of the present disclosure at lowerstringency hybridization conditions. Changes in the stringency ofhybridization and signal detection are primarily accomplished throughthe manipulation of formamide concentration (lower percentages offormamide result in lowered stringency); salt conditions, ortemperature. For example, lower stringency conditions include anovernight incubation at 37° C. in a solution comprising 6×SSPE(20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA, pH 7.4), 0.5% SDS, 30%formamide, 100 μg/ml salmon sperm blocking DNA; followed by washes at50° C. with 1×SSPE, 0.1% SDS. In addition, to achieve even lowerstringency, washes performed following stringent hybridization can bedone at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

As used herein, “percent identity” of two amino acids or of two nucleicacids is determined using the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87:2264.2268, 1990), modified as in Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score 100,wordlength=12, to determine nucleotide sequences identity to the nucleicacid molecules of the disclosure. BLAST protein searches are performedwith the XBLAST program, score=50, wordlength=3, to determine an aminoacid sequence identity to a polypeptide of the disclosure. To obtaingapped alignments for comparison purposes, Gapped BLAST is utilized asdescribed in Altschul et al. (Nucleic Acids. Res. 25:3389-3402, 1997).When utilizing BLAST and Gapped BLAST programs, the default parametersof the respective programs (e.g., XBLAST and NBLAST) are used.

Further embodiments of the present disclosure include polynucleotidesencoding laminin-521 chain polypeptides having at least 70% identity,preferably at least 80% identity, and most preferably at least 90%identity to one or more of the polypeptide sequences contained in SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

As used herein, “α5 polynucleotide” refers to polynucleotides encoding alaminin α5 chain. Such polynucleotides can be characterized by one ormore of the following: (a) polynucleotides that encode polypeptideswhich share at least 70% identity, preferably 80% identity, and mostpreferably at least 90% identity with a sequence selected of SEQ ID NO:5; (b) polynucleotides that hybridize under low or high stringencyconditions to the coding sequence of SEQ ID NO: 5 or complementarysequences thereof; or (c) polynucleotides encoding a laminin α5 chainpolypeptide with a general structure selected from the group consistingof R1-R2-R3, R1-R2-R4, R3, R4, R1-R3, R1-R4, R2-R3, and R2-R4, whereinR1 and R2 are as described above, R3 is a secreted α5 chain, and R4 is asecreted α5 chain that comprises an epitope tag.

As used herein, “β2 polynucleotides” refers to polynucleotides encodinga β2 laminin chain of the same name. Such polynucleotides can becharacterized by one or more of the following: (a) polynucleotides thatencode polypeptides which share at least 70% identity, preferably atleast 80%, and most preferably at least 90% identity with the sequenceof SEQ ID NO: 4; (b) polynucleotides that hybridize under low or highstringency conditions to the coding sequences of SEQ ID NO: 4, orcomplementary sequences thereof; or (c) polynucleotides encoding apolypeptide with a general structure selected from R1-R2-R3, R1-R2-R4,R3, R4, R1-R3, R1-R4, R2-R3, and R2-R4, wherein R1 and R2 are asdescribed above, R3 is a secreted β2 chain, and R4 is a secreted β2chain that comprises an epitope tag.

As used herein, “γ1 polynucleotides” refers to polynucleotides encodinga γ1 laminin chain of the same name. Such polynucleotides can becharacterized by one or more of the following: (a) polynucleotides thatencode polypeptides which share at least 70% identity, preferably atleast 80%, and most preferably at least 90% identity with the sequenceof SEQ ID NO: 6; (b) polynucleotides that hybridize under low or highstringency conditions to the coding sequence of SEQ ID NO: 6 orcomplementary sequences thereof; or (c) polynucleotides that encode apolypeptide with a general structure selected from R1-R2-R3, R1-R2-R4,R3, R4, R1-R3, R1-R4, R2-R3, and R2-R4, wherein R1 and R2 are asdescribed above, R3 is a secreted γ1 chain, and R4 is a secreted γ1chain that comprises an epitope tag.

As used herein, the term “epitope tag” refers to a polypeptide sequencethat is expressed as part of a chimeric protein, where the epitope tagserves as a recognition site for binding of antibodies generated againstthe epitope tag, or for binding of other molecules that can be used foraffinity purification of sequences containing the tag.

In preferred embodiments, cDNAs encoding the laminin α5, β2 and γ1chains, or fragments thereof, are subcloned into an expression vector.Alternatively, laminin α5, β2 and/or γ1 gene sequences, including one ormore introns, can be used for sub-cloning into an expression vector.

In other aspects, the present disclosure provides laminin-521expressing-cells that have been transfected with an expression vectorcontaining promoter sequences that are operatively linked to nucleicacid sequences encoding at least one polypeptide sequence comprising asequence selected from the group consisting of the α5, β2 and γ1 chainsof laminin-521, wherein the transfected cells secrete heterotrimericlaminin-521 containing the recombinant laminin chain. In preferredembodiments, the cells are systematically transfected with recombinantexpression vectors containing promoter sequences that are operativelylinked to nucleic acid sequences encoding polypeptide sequencescomprising the α5, β2 and γ1 chains of laminin-521, which are even morepreferably all human chains. After the multiple transfections, the cellsexpress recombinant laminin-521 chains, which form the heterotrimericr-laminin-521.

Transfection of the expression vectors into eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to calcium phosphate co-precipitation, electroporation, orliposome mediated-, DEAE dextran mediated-, polycationic mediated-, orviral mediated transfection. Transfection of bacterial cells can be doneby standard methods.

In preferred embodiments, the cells are stably transfected. Methods forstable transfection and selection of appropriate transfected cells areknown in the art. In other preferred embodiments, a CMV promoter drivenexpression vector is used in a human kidney embryonic 293 cell line.

Any cell capable of expressing and secreting the r-laminin-521 can beused. Preferably, eukaryotic cells are used, and most preferablymammalian cells are used, including but not limited to kidney andepithelial cell lines. The promoter sequence used to drive expression ofthe individual chains or r-laminin-521 may be constitutive (driven byany of a variety of promoters, including but not limited to, CMV, SV40,RSV, actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive). Carbohydrate and disulfide post-translationalmodifications are believed to be required for laminin-521 proteinfolding and function. This makes the use of eukaryotic cells preferablefor producing functional r-laminin-521, although other systems areuseful for obtaining, for example, antigens for antibody production. Inmost preferred embodiments, the mammalian cells do not express thelaminin β2 chain endogenously. In other preferred embodiments, the cellsdo not express all of the laminin-521 chains endogenously.

The protein may comprise additional sequences useful for promotingpurification of the protein, such as epitope tags and transport signals.Examples of such epitope tags include, but are not limited to FLAG(Sigma Chemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad,Calif.), 6-His (Invitrogen; Novagen, Madison, Wis.), and HA (BoehringerManheim Biochemicals). Examples of such transport signals include, butare not limited to, export signals, secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In some embodiments, at least one of the laminin chain polypeptidesequences, or fragments thereof, is operatively linked to a nucleic acidsequence encoding an “epitope tag”, so that at least one of the chainsis expressed as a fusion protein with an expressed epitope tag. Theepitope tag may be expressed as the amino terminus, the carboxyterminus, or internal to any of the polypeptide chains comprisingr-laminin-521, so long as the resulting r-laminin-521 remainsfunctional.

In other embodiments, one of the r-laminin-521 chains is expressed as afusion protein with a first epitope tag, and at least one otherr-laminin chain is expressed as a fusion protein with a second differentepitope tag. This permits multiple rounds of purification to be carriedout. Alternatively, the same epitope tag can be used to create fusionproteins with more than one of the r-laminin chains.

In further embodiments, the epitope tag can be engineered to becleavable from the r-laminin-521 chain(s). Alternatively, no epitope tagis fused to any of the r-laminin-521 chains, and the r-laminin-521 isisolated by standard techniques, including but not limited to affinitychromatography using laminin-521 specific antibodies or otherlaminin-521 binding molecules.

Media from cells transfected with a single laminin chain are initiallyanalyzed on Western blots using laminin chain-specific antibodies. Theexpression of single laminin chains following transfection is generallyintracellular. Clones showing reactivity against individual transfectedchain(s) are verified by any appropriate method, such as PCR, reversetranscription-PCR, or nucleic acid hybridization, to confirmincorporation of the transfected gene. Preferably, analysis of genomicDNA preparations from such clones is done by PCR using lamininchain-specific primer pairs. Media from transfected clones producing allthree chains are further analyzed for r-laminin-521 secretion and/oractivity, by any appropriate method, including Western blot analysis andcell binding assays.

In preferred embodiments, purification of r-laminin-521 is accomplishedby passing media from the transfected cells through an antibody affinitycolumn. In some embodiments, antibodies against a peptide epitopeexpressed on at least one of the recombinant chains are attached to anaffinity column, and bind the r-laminin-521 that has been secreted intothe media. The r-laminin-521 is removed from the column by passingexcess peptide over the column. Eluted fractions are analyzed by anyappropriate method, including gel electrophoresis and Western blotanalysis. In further embodiments, the peptide epitope can be cleavedafter purification. In other embodiments, two or three separater-laminin chains are expressed as fusion proteins, each with a differentepitope tag, permitting two or three rounds of purification and a doublyor triply isolated r-laminin-521. The epitope tag can be engineered soas to be cleavable from the r-laminin-521 chain(s) after purification.Alternatively, no epitope tag is fused to any of the r-laminin-521chains, and the r-laminin-521 is isolated by standard techniques,including but not limited to affinity chromatography using laminin-521specific antibodies or other laminin-521 binding molecules.

In other embodiments, purification of r-laminin-521 is accomplished bypassing media from the transfected cells through a gel-filtrationchromatography column. Eluted fractions are analyzed by any appropriatemethod, including gel electrophoresis and Western blot analysis.Fractions containing r-laminin-521 are collected and purity of thespecimen is evaluated by any appropriate method, including gelelectrophoresis and Western blot analysis. In some embodiments, theprotein solution can be passed through a gel-filtration chromatographycolumn again to gain higher purity of the protein. In some embodiments,to achieve higher purity of r-laminin-521 solution, the media orr-laminin-521 solution from the previous purification steps can bepassed through an ion-exchange column. Eluted fractions are analyzed byany appropriate method, including gel electrophoresis and Western blotanalysis. Fractions containing r-laminin-521 are collected and purity ofthe specimen is evaluated by any appropriate method, including mentionedabove.

The laminin-521 polypeptide chains of the present disclosure alsoinclude (i) substitutions with one or more of the non-conserved aminoacid residues, where the substituted amino acid residues may or may notbe one encoded by the genetic code, or (ii) substitution with one ormore amino acid residues having substituent groups, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

In particular embodiments, the isolated laminin-521 comprises threechains. The first chain comprises a polypeptide with at least 80%identity to a polypeptide sequence of SEQ ID NO: 1 (i.e. the α5 lamininchain). The second chain comprises a polypeptide with at least 70%identity to a polypeptide sequence of SEQ ID NO: 2 (i.e. the β2 lamininchain). The third chain comprises a polypeptide with at least 70%identity to a polypeptide sequence of SEQ ID NO: 3 (i.e. the γ1 lamininchain). These first, second, and third chains are assembled intorecombinant laminin-521.

In more specific embodiments, the polypeptide of the first chain has atleast 80% identity to the polypeptide sequence of SEQ ID NO: 1, thepolypeptide of the second chain has at least 80% identity to thepolypeptide sequence of SEQ ID NO: 2, and the polypeptide of the thirdchain has at least 80% identity to the polypeptide sequence of SEQ IDNO: 3.

In more specific embodiments, the polypeptide of the first chain has atleast 90% identity to the polypeptide sequence of SEQ ID NO: 1, thepolypeptide of the second chain has at least 90% identity to thepolypeptide sequence of SEQ ID NO: 2, and the polypeptide of the thirdchain has at least 90% identity to the polypeptide sequence of SEQ IDNO: 3.

In particular embodiments, the first chain comprises the polypeptidesequence of SEQ ID NO: 1, the second chain comprises the polypeptidesequence of SEQ ID NO: 2, and the third chain comprises the polypeptidesequence of SEQ ID NO: 3.

In particular embodiments, the first chain is the polypeptide sequenceof SEQ ID NO: 1, the second chain is the polypeptide sequence of SEQ IDNO: 2, and the third chain is the polypeptide sequence of SEQ ID NO: 3.

The present disclosure further provides pharmaceutical compositionscomprising isolated laminin-521 and a pharmaceutically acceptablecarrier. In preferred embodiments, the pharmaceutical compositioncomprises isolated r-laminin-521. According to these aspects of thedisclosure, other agents can be included in the pharmaceuticalcompositions, depending on the condition being treated. Thepharmaceutical composition may further comprise one or more othercompounds, including but not limited to any of the collagens, otherlaminin types, fibronectin, vitronectin, cadherins, integrins,α-dystroglycan, entactin/nidogen, α-dystroglycan, glycoproteins,proteoglycans, heparan sulfate proteoglycan, glycosaminoglycans,epidermal growth factor, vascular endothelial growth factor, fibroblastgrowth factor, or nerve growth factors, and peptide fragments thereof.

Pharmaceutical preparations comprising isolated laminin-521 can beprepared in any suitable form, and generally comprise the isolatedlaminin-521 in combination with a pharmaceutically acceptable carrier.The carriers can be injectable carriers, topical carriers, transdermalcarriers, and the like. The preparation may advantageously be in a formfor topical administration, such as an ointment, gel, cream, spray,dispersion, suspension or paste. The preparations may furtheradvantageously include preservatives, antibacterials, antifingals,antioxidants, osmotic agents, and similar materials in composition andquantity as is conventional. Suitable solutions for use in accordancewith the disclosure are sterile, are not harmful for the proposedapplication, and may be subjected to conventional pharmaceuticaloperations such as sterilization and/or may contain conventionaladjuvants, such as preservatives, stabilizers, wetting agents,emulsifiers, buffers etc. For assistance in formulating the compositionsof the present disclosure, one may refer to Remington's PharmaceuticalSciences, 15th Ed., Mack Publishing Co., Easton, Pa. (1975).

In further aspects, the present disclosure comprises medical deviceswith improved biocompatibility, wherein the exterior surfaces of thedevices are coated with isolated laminin-521 or pharmaceuticalcompositions thereof, alone or in combination with other proteins oragents that serve to increase the biocompatibility of the devicesurface. The coated device stimulates cell attachment (such asendothelial cell attachment), and provides for diminished inflammationand/or infection at the site of entry of the device.

Such medical devices can be of any material used for implantation intothe body, and preferably are made of or coated with a biocompatiblemetal, such as stainless steel or titanium. Alternatively, the device ismade of or coated with a ceramic material or a polymer, such aspolyester, polyglycolic acid, or a polygalactose-polyglycolic acidcopolymer.

If the device is made of a natural or synthetic biodegradable materialin the form of a mesh, sheet or fabric, isolated laminin-521 orpharmaceutical compositions thereof may be applied directly to thesurface thereof. Appropriate cells may then be cultured on the matrix toform transplantable or implantable devices, including dental abutmentpieces, needles, metal pins or rods, indwelling catheters, colostomytubes, surgical meshes and any other appliance for which coating withisolated laminin-521 is desirable. Alternatively, the devices may beimplanted and cells may be permitted to attach in vivo.

Coupling of the isolated laminin-521 may be non-covalent (such as byadsorption), or by covalent means. The device may be immersed in,incubated in, or sprayed with the isolated laminin-521 or pharmaceuticalcompositions thereof.

The dosage regimen for various treatments using the isolated laminin-521of the present disclosure is based on a variety of factors, includingthe type of injury or condition, the age, weight, sex, medical conditionof the individual, the severity of the condition, and the route ofadministration. Thus, the dosage regimen may vary widely, but can bedetermined routinely by a physician using standard methods. Laminins areextremely potent molecules, and one or a few molecules per cell couldproduce an effect. Thus, effective doses in the pico-gram per milliliterrange are possible if the delivery is optimized.

In other aspects, the present disclosure provides a new material,isolated laminin-521, which permits human embryonic stem cell survivalafter dissociation into single cell suspension. The stem cells weredissociated into single cell suspension by Trypsin-EDTA treatment,pelleted by centrifugation, resuspended into O3 medium, filtered througha 40 μm sieve, and plated at a density of 30 Kcells/cm² on cell culturedishes precoated by either isolated laminin-521, laminin-511, orMatrigel. After one day in culture, the cells plated on Matrigel died,as is known in the art. The human embryonic stem cells plated onlaminin-521 and in most cases on laminin-511 survived and started toproliferate, forming small colonies of pluripotent cells.

In further aspects, the present disclosure provides a method to expandhuman embryonic stem cells in pluripotent state. It has been shown thathuman embryonic stem cells plated in single cell suspension onlaminin-521 survived and proliferated at a higher rate than that ofclassical methods known in the art. After 3 passages (1 month) the cellspassaged in a single cell suspension underwent the same number of celldivisions as that of cell cultures passaged in pieces on Matrigel after20 passages (3 months). Therefore, the new method was advantageous interms of time and labor, which may provide significant economicalprofits.

Laminin-521 is normally expressed and secreted by human pluripotentembryonic stem cells and can also be found in the kidneys, neuromuscularjunctions, lungs, and placenta.

The availability of pure laminin-521 would enable studies of the effectsof the protein on cellular differentiation and maintenance of cellularphenotypes. Thus, numerous research and therapeutic purposes including,but not limited to, treating injuries to tissues, promoting cellattachment, expansion and migration, ex vivo cell therapy, improving thebiocompatibility of medical devices, and preparing improved cell culturedevices and media, would be furthered if pure intact isolatedlaminin-521 were available. Also, the effects of pure laminin-521 onstem cells would be important to study as this protein is expressed inthe early mammalian embryo.

Thus, there is a need in the art for isolated laminin-521 for researchand therapeutic purposes, and methods for making isolated laminin-521.Laminin-521 can serve as a matrix for long-term self-renewal and fastmultiplication of dissociated human ES and induced pluripotent stem(IPS) cells under completely chemically defined, feeder-free, andanimal-protein-free (xeno-free) conditions. A LN-521 based system iseasy to use and easy to automate, and allows fast scale-up of humanES/IPS cultures.

The present disclosure also relates to a cell culture medium that can beused to provide nutrition to cells, particularly stem cells. In thisregard, stem cells typically require two things to be cultured: (1) asubstrate or coating that provides a structural support for the stemcell; and (2) a cell culture medium to provide nutrition to the stemcell. The substrate or coating (1) is generally placed on, for example,a petri dish or some other container.

As used herein, the term “self-renewal” refers to the ability of thestem cell to go through numerous cycles of cell division and remainundifferentiated (i.e. pluripotent). Pluripotency itself refers to theability of the stem cell to differentiate into any cell type. The term“proliferation” refers to the ability of the stem cell to divide.Survival refers to the ability of the stem cell to live, whetherdifferentiated or undifferentiated, and does not require the stem cellto maintain its ability to divide or to differentiate.

The cell culture medium of the present disclosure is particularlysuitable for being used with a substrate that contains laminin-521and/or laminin-511. These laminins activate α6β1 integrins, which inturn leads to activation of the PI3K/Akt pathway. This increases thepluripotency, self-renewal, and/or proliferation of the stem cells. Itis contemplated that the substrate may consist of laminin-521 orlaminin-511, either intact, as separate chains, or as fragments thereof.Recombinant laminin-521 and recombinant laminin-511 are commerciallyavailable. Many different molecules can activate the PI3K/Akt pathway,though with different efficiencies. For example, TGF beta 1 and bFGFactivate this pathway. The use of laminin-521 and/or laminin-511 allowsthe quantity of such molecules to be reduced in the cell culture medium.Laminin-521 conveys the highest dose of signal via α6β1 integrin,activating the PI3K/Akt pathway. The use of laminin-521 allows forsingle-cell suspension passaging without the addition ofcell-detrimental rho-kinase (ROCK) inhibitor to increase cell survivalafter single-cell enzymatic dissociation. Previously, single-cellenzymatic passage of human ES cells without using artificial apoptosisinhibitors was impossible. The simplicity of the passaging proceduremeans the experimental variance is reduced and allows the process to beautomated for high-throughput cell culture and results without theextensive training and costs of cell culture staff. In addition, humanES and iPS cells plated on laminin-521 or laminin-511 grow as amonolayer, which makes the culture homogeneous since cells are equallyexposed to the matrix and the cell culture medium. Such human ES cellcultures, grown in a chemically defined, xeno-free environment onlaminin-521, passaged as single cells in the absence of ROCK inhibitorexpand continuously for months at an even better growth rate compared tocells grown on Matrigel passaged as clumps. These pluripotent long-termexpanded cells homogeneously express Oct4 and remain karyotypicallynormal. Thus, one can obtain human ES and iPS cells with sustainedsurvival and proliferation capacity.

The average contact area and spreading homogeneity is much larger forcells cultured on laminin-511 compared to other available substrata.Human ES cells grown on laminin-511 over 3 months maintain pluripotencyand can generate teratomas after engraftment into SCID mice. Laminin-511also supports the self-renewal of mouse ES cells for over 5 monthswithout the presence of LIF or feeder cells, when other known matricesare unable to do so for longer than a couple of weeks.

The stem cells to be grown with this cell culture medium can be inducedpluripotent stem cells, embryonic stem cells, adult stem cells, fetalstem cells, amniotic stem cells, and generally any pluripotent stemcell.

Typically, cell culture media include a large number and a large amountof various growth factors and cytokines to inhibit differentiation andimprove proliferation. One advantage of the cell culture medium of thepresent disclosure is that it does not contain as many growth factors orcytokines, or such high amounts.

Most generally, the cell culture medium of the present disclosurerequires lower amounts of basic fibroblast growth factor (bFGF) thantypically used. It is contemplated that the cell culture medium maycomprise from greater than zero to 3.9 nanograms per milliliter (ng/mL)of bFGF. The bFGF is human bFGF so that the cell culture medium istotally human and defined. In some more specific embodiments, the cellculture medium may comprise 3.5 or lower ng/mL of bFGF. In otherembodiments, the cell culture medium may comprise from 0.5 to 3.5 ng/mLof bFGF. In some embodiments, the cell culture medium may have zerobFGF, i.e. no bFGF is present.

Generally, the cell culture medium includes a liquid phase in which atleast one inorganic salt, at least one trace mineral, at least oneenergy substrate, at least one lipid, at least one amino acid, at leastone vitamin, and at least one growth factor (besides bFGF) aredissolved. Table 1 below includes a list of various such ingredientswhich may be present in the cell culture medium of the presentdisclosure, and the minimum and maximum concentrations if the ingredientis present. The values are presented in scientific notation. Forexample, “4.1E-01” should be interpreted as 4.1×10⁻⁰¹.

TABLE 1 molar Min. Max. Min. Max. mass Conc. Conc. Conc. Conc.Ingredient (g/mol) (mM) (mM) (ng/mL) (ng/mL) INORGANIC SALTS Calciumchloride (Anhydrous) 110.98 4.1E−01 1.6E+00 4.6E+04 1.8E+05 HEPES 238.35.9E+00 1.8E+01 1.4E+06 4.2E+06 Lithium Chloride (LiCl) 42.39 4.9E−011.5E+00 2.1E+04 6.2E+04 Magnesium chloride (Anhydrous) 95.21 1.2E−013.6E−01 1.1E+04 3.4E+04 Magnesium Sulfate (MgSO₄) 120.37 1.6E−01 4.8E−011.9E+04 5.8E+04 Potassium chloride (KCl) 74.55 1.6E+00 4.9E+00 1.2E+053.6E+05 Sodium bicarbonate (NaHCO₃) 84.01 9.0E+00 4.4E+01 7.6E+053.7E+06 Sodium chloride (NaCl) 58.44 4.7E+01 1.4E+02 2.8E+06 8.3E+06Sodium phosphate, 141.96 2.0E−01 5.9E−01 2.8E+04 8.3E+04 dibasic(Anhydrous) Sodium phosphate, monobasic 137.99 1.8E−01 5.3E−01 2.4E+047.3E+04 monohydrate (NaH₂PO₄—H₂O) TRACE MINERALS Ferric Nitrate(Fe(NO₃)₃—9H₂O) 404 4.9E−05 1.9E−04 2.0E+01 7.5E+01 Ferrous sulfateheptahydrate 278.01 5.9E−04 1.8E−03 1.6E+02 4.9E+02 (FeSO₄—7H₂O)Copper(II) sulfate 249.69 2.0E−06 8.0E−06 5.1E−01 2.0E+00 pentahydrate(CuSO₄—5H₂O) Zinc sulfate heptahydrate 287.56 5.9E−04 1.8E−03 1.7E+025.1E+02 (ZnSO₄—7H₂O) Ammonium Metavanadate NH₄VO₃ 116.98 5.5E−06 1.6E−056.4E−01 1.9E+00 Manganese Sulfate 169.02 9.9E−07 3.0E−06 1.7E−01 5.0E−01monohydrate (MnSO₄—H₂O) NiSO₄—6H₂O 262.85 4.9E−07 1.5E−06 1.3E−013.8E−01 Selenium 78.96 8.9E−05 2.7E−04 7.0E+00 2.1E+01 Sodium MetaSilicate 284.2 4.8E−04 1.4E−03 1.4E+02 4.1E+02 Na₂SiO₃—9H₂O SnCl₂ 189.626.2E−07 1.9E−06 1.2E−01 3.5E−01 Molybdic Acid, Ammonium salt 1235.869.9E−07 3.0E−06 1.2E+00 3.7E+00 CdCl₂ 183.32 6.1E−06 1.8E−05 1.1E+003.4E+00 CrCl₃ 158.36 9.9E−07 3.0E−06 1.6E−01 4.7E−01 AgNO₃ 169.874.9E−07 1.5E−06 8.3E−02 2.5E−01 AlCl₃—6H₂O 241.43 2.4E−06 7.3E−065.9E−01 1.8E+00 Barium Acetate (Ba(C₂H₃O₂)₂) 255.42 4.9E−06 1.5E−051.3E+00 3.8E+00 CoCl₂—6H₂O 237.93 4.9E−06 1.5E−05 1.2E+00 3.5E+00 GeO₂104.64 2.5E−06 7.5E−06 2.6E−01 7.8E−01 KBr 119 4.9E−07 1.5E−06 5.9E−021.8E−01 Kl 166 5.0E−07 1.5E−06 8.3E−02 2.5E−01 NaF 41.99 4.9E−05 1.5E−042.1E+00 6.2E+00 RbCl 120.92 4.9E−06 1.5E−05 5.9E−01 1.8E+00 ZrOCl₂—8H₂O178.13 4.9E−06 1.5E−05 8.7E−01 2.6E+00 ENERGY SUBSTRATES D-Glucose180.16 6.9E+00 2.1E+01 1.2E+06 3.7E+06 Sodium Pyruvate 110.04 2.0E−015.9E−01 2.2E+04 6.5E+04 LIPIDS Linoleic Acid 280.45 9.4E−05 2.8E−042.6E+01 7.9E+01 Lipoic Acid 206.33 2.0E−04 7.8E−04 4.1E+01 1.6E+02Arachidonic Acid 304.47 6.5E−06 1.9E−05 2.0E+00 5.9E+00 Cholesterol386.65 5.6E−04 1.7E−03 2.2E+02 6.5E+02 DL-alpha tocopherol-acetate472.74 1.5E−04 4.4E−04 6.9E+01 2.1E+02 Linolenic Acid 278.43 3.5E−051.0E−04 9.7E+00 2.9E+01 Myristic Acid 228.37 4.3E−05 1.3E−04 9.8E+002.9E+01 Oleic Acid 282.46 3.5E−05 1.0E−04 9.8E+00 2.9E+01 Palmitic Acid256.42 3.8E−05 1.1E−04 9.8E+00 2.9E+01 Palmitoleic acid 254.408 3.9E−051.2E−04 9.8E+00 2.9E+01 Stearic Acid 284.48 3.4E−05 1.0E−04 9.8E+002.9E+01 AMINO ACIDS L-Alanine 89.09 2.5E−02 2.1E−01 2.2E+03 1.8E+04L-Arginine hydrochloride 147.2 2.7E−01 1.5E+00 4.0E+04 2.2E+05L-Asparagine-H₂O 150.13 5.0E−02 2.1E−01 7.5E+03 3.1E+04 L-Aspartic acid133.1 2.5E−02 2.1E−01 3.3E+03 2.7E+04 L-Cysteine-HCl—H₂O 175.63 3.9E−021.2E−01 6.9E+03 2.1E+04 L-Cystine dihydrochloride 313.22 3.9E−02 1.2E−011.2E+04 3.7E+04 L-Glutamic acid 147.13 2.5E−02 2.1E−01 3.7E+03 3.0E+04L-Glutamine 146.15 1.5E+00 4.4E+00 2.1E+05 6.4E+05 Glycine 75.07 1.5E−014.4E−01 1.1E+04 3.3E+04 L-Histidine monohydrochloride 209.63 5.9E−021.8E−01 1.2E+04 3.7E+04 monohydrate L-Isoleucine 131.17 1.6E−01 4.9E−012.1E+04 6.4E+04 L-Leucine 131.17 1.8E−01 5.3E−01 2.3E+04 7.0E+04L-Lysine hydrochloride 182.65 2.0E−01 5.9E−01 3.6E+04 1.1E+05L-Methionine 149.21 4.5E−02 1.4E−01 6.8E+03 2.0E+04 L-Phenylalanine165.19 8.5E−02 2.5E−01 1.4E+04 4.2E+04 L-Proline 115.13 1.1E−01 3.2E−011.2E+04 3.7E+04 L-Serine 105.09 1.5E−01 4.4E−01 1.5E+04 4.6E+04L-Threonine 119.12 1.8E−01 5.3E−01 2.1E+04 6.3E+04 L-Tryptophan 204.231.7E−02 5.2E−02 3.5E+03 1.1E+04 L-Tyrosine disodium 225.15 8.4E−023.7E−01 1.9E+04 8.4E+04 salt hydrate L-Valine 117.15 1.8E−01 5.3E−012.1E+04 6.2E+04 VITAMINS Ascorbic acid 176.12 1.3E−01 3.8E−01 2.2E+046.7E+04 Biotin 244.31 5.6E−06 1.7E−05 1.4E+00 4.1E+00 B₁₂ 1355.372.0E−04 5.9E−04 2.7E+02 8.0E+02 Choline chloride 139.62 2.5E−02 7.5E−023.5E+03 1.1E+04 D-Calcium pantothenate 238.27 1.8E−03 1.4E−02 4.4E+023.4E+03 Folic acid 441.4 2.4E−03 7.1E−03 1.0E+03 3.1E+03 i-Inositol180.16 2.7E−02 1.1E−01 4.9E+03 1.9E+04 Niacinamide 122.12 6.5E−032.0E−02 7.9E+02 2.4E+03 Pyridoxine hydrochloride 205.64 3.8E−03 1.1E−027.8E+02 2.4E+03 Riboflavin 376.36 2.3E−04 6.8E−04 8.6E+01 2.6E+02Thiamine hydrochloride 337.27 3.3E−03 3.6E−02 1.1E+03 1.2E+04 GROWTHFACTORS/PROTEINS GABA 103.12 0 1.5E+00 0 1.5E+05 Pipecolic Acid 129 01.5E−03 0 1.9E+02 bFGF 18000 0 2.17E−07  0 3.9E+00 TGF beta 1 25000 03.5E−08 0 8.8E−01 Human Insulin 5808 0 5.9E−03 0 3.4E+04 HumanHolo-Transferrin 78500 0 2.1E−04 0 1.6E+04 Human Serum Albumin 67000 02.9E−01 0 2.0E+07 Glutathione (reduced) 307.32 0 9.6E−03 0 2.9E+03 OTHERCOMPONENTS Hypoxanthine Na 136.11 5.9E−03 2.6E−02 8.0E+02 3.6E+03 Phenolred 354.38 8.5E−03 2.5E−02 3.0E+03 9.0E+03 Putrescine-2HCl 161.072.0E−04 5.9E−04 3.2E+01 9.5E+01 Thymidine 242.229 5.9E−04 1.8E−031.4E+02 4.3E+02 2-mercaptoethanol 78.13 4.9E−02 1.5E−01 3.8E+03 1.1E+04Pluronic F-68 8400 1.2E−02 3.5E−02 9.8E+04 2.9E+05 Tween 80 1310 1.6E−044.9E−04 2.2E+02 6.5E+02

The liquid phase of the cell culture medium may be water, serum, oralbumin.

Many of the ingredients or components listed above in Table 1 are notnecessary, or can be used in lower concentrations.

It is contemplated that the cell culture medium may contain insulin oran insulin substitute. Similarly, the cell culture medium may containtransferrin or a transferrin substitute.

In more specific embodiments, it is contemplated that the cell culturemedium may not (1) contain albumin, (2) insulin or insulin substitute,(3) transferrin or transferrin substitute, or any combination of thesethree components.

It should be noted that other cell culture mediums may contain growthfactors such as interleukin-1 beta (IL-β or catabolin), interleukin-6(IL6), or pigment epithelium derived factor (PEDF). Such growth factorsare not present in the cell culture medium of the present disclosure.

One specific formula for a cell culture medium is provided in Table 2:

TABLE 2 Ingredient Amount Unit bFGF 0.39 microgram (μg) Albumin 1.34milligram (mg) Insulin 2 mg Lithium Chloride 4.23 mg GABA 0.01 mg TGFbeta 1 0.06 μg Pipecolic acid 0.013 mg L-glutamine 2.92 grams MEMnon-essential amino acid solution 1 mL DMEM/F12 100 mL

In this regard, MEM non-essential amino acid solution is typicallyprovided in a 100× concentrate. The MEM of Table 2 is used afterdilution back to 1×, and contains the following amino acids in thefollowing concentration listed in Table 3:

TABLE 3 MEM Amino Acids Concentration (ng/mL) Glycine 7.50E+03 L-Alanine8.90E+03 L-Asparagine 1.32E+04 L-Aspartic acid 1.33E+04 L-Proline1.15E+04 L-Serine 1.05E+04

DMEM/F12 contains the following ingredients listed in Table 4:

TABLE 4 Concentration DMEM/F12 Ingredients (ng/mL) Glycine 187.5L-Alanine 44.5 L-Arginine hydrochloride 1475 L-Asparagine-H₂O 75L-Aspartic acid 66.5 L-Cysteine hydrochloride-H₂O 175.6 L-Cystine 2HCl312.9 L-Glutamic Acid 73.5 L-Glutamine 3650 L-Histidinehydrochloride-H₂O 314.8 L-Isoleucine 544.7 L-Leucine 590.5 L-Lysinehydrochloride 912.5 L-Methionine 172.4 L-Phenylalanine 354.8 L-Proline172.5 L-Serine 262.5 L-Threonine 534.5 L-Tryptophan 90.2 L-Tyrosinedisodium salt dihydrate 557.9 L-Valine 528.5 Biotin 0.035 Cholinechloride 89.8 D-Calcium pantothenate 22.4 Folic Acid 26.5 Niacinamide20.2 Pyridoxine hydrochloride 20 Riboflavin 2.19 Thiamine hydrochloride21.7 Vitamin B₁₂ 6.8 i-Inositol 126 Calcium Chloride (CaCl₂) (anhyd.)1166 Cupric sulfate (CuSO₄—5H₂O) 0.013 Ferric Nitrate (Fe(NO₃)₃—9H₂O)0.5 Ferric sulfate (FeSO₄—7H₂O) 4.17 Magnesium Chloride (anhydrous)286.4 Magnesium Sulfate (MgSO₄) (anhyd.) 488.4 Potassium Chloride (KCl)3118 Sodium Bicarbonate (NaHCO₃) 24380 Sodium Chloride (NaCl) 69955Sodium Phosphate dibasic 710.2 (Na₂HPO₄) anhydrous Sodium Phosphatemonobasic 625 (NaH₂PO₄—H₂O) Zinc sulfate (ZnSO₄—7H₂O) 4.32 D-Glucose(Dextrose) 31510 Hypoxanthine Na 23.9 Linoleic Acid 0.42 Lipoic Acid1.05 Phenol Red 81 Putrescine 2HCl 0.81 Sodium Pyruvate 550 Thymidine3.65

The combination of the laminin substrate with the cell culture medium ofthe present disclosure results in a cell culture system that can becheaper, yet provides higher efficiency in maintaining pluripotent stemcells. In essence, all that is required is a laminin and a minimalamount of nutrition. It is particularly contemplated that the lamininused in combination with this cell culture medium is either LN-511 orLN-521.

The following examples are for purposes of further illustrating thepresent disclosure. The examples are merely illustrative and are notintended to limit devices made in accordance with the disclosure to thematerials, conditions, or process parameters set forth therein.

EXAMPLES Example 1

Cloning of the Human Laminin β2 cDNA

The 5.6 kb fragment of human laminin β2 cDNA was PCR-amplified fromhuman liver cDNA library (BD Biosciences) using primers5′-GTGGTACCCACAGGCAGAGTTGAC-3′ (SEQ ID NO: 7) and5′-GCTCTAGAGCTCTTCAGTGCATAGGC-3′ (SEQ ID NO: 8) thus introducingartificial XbaI and KpnI cutting sites on the ends of the fragment. Todecrease the error rate during the PCR amplification, Phusion™high-fidelity PCR Kit (Finnzymes) was used. Subsequently, the fragmentwas digested with XbaI and KpnI and subcloned into pSK vector digestedwith the same restriction endonucleases (pSKHLAMB2 plasmid). To verifythe integrity of the sequence, several clones of pSKHLAMB2 plasmid weresequenced. Sequencing was performed on an ABI PRISM™ 310 GeneticAnalyzer (Perkin Elmer) using ABI PRISM® BigDye™ Terminator CycleSequencing kit (PE Applied Biosystems). Only complete matches with theNCBI database human laminin 62 sequence were selected for furthercloning.

Expression Constructs

For expression of the human laminin β2 chain pSKHLAMB2 plasmid wasdigested with XbaI and KpnI and subcloned into XbaI-KpnI treated pcDNA3.1(+) vector (Invitrogen).

The constructs used for expression of human laminin α5 (HLN5Full.pcDNAconstruct) and γ1 (HG1 construct) have been described previously (Doi,M. et al., J. Biol. Chem. 277(15), 12741-8 (2002)).

Antibodies

Anti-laminin β2 (MAB2066) monoclonal antibody (mAb) was purchased fromR@D Systems. Anti-laminin α5 mAb (2F7) was purchased from Abnova.Anti-laminin β1 mAb (MAB1921) was purchased from Chemicon. Anti-lamininγ1 (H-190) rabbit polyclonal antibody was purchased from Santa CruzBiotechnology, Inc.

Production and Purification of Recombinant Laminin-521

r-laminin-521 was produced in human embryonic kidney cells (HEK293, ATCCCRL-1573) cultured in DMEM, 10% FCS in humidified 5% CO₂ atmosphere at37° C. Wild-type cells were transfected using the standardcalcium-phosphate method with the HG1 construct and stable colonies wereselected using 100 mg/ml hygromycin (Cayla). All further cell cultureand clonal expansion was carried out in continuous presence of relevantselection antibiotics. A highly expressing clone was then transfectedwith the human laminin β2 construct and stable clones were selectedusing 500 mg/ml G418 (Life Technologies). A clone highly expressing bothlaminin γ1 and laminin β2 was finally transfected with theHLN5Full.pcDNA construct and stable colonies were selected using 200mg/ml zeocin (Cayla). The clones showing the highest secretion wereexpanded further.

For production of r-laminin-521, confluent cells were cultured in DMEMfor up to five days. r-laminin-521 was affinity purified using anti-FLAGM2 matrix (Sigma). The collected medium was incubated in batch mode withthe matrix overnight at 4° C. with agitation. Bound r-laminin-521 wascompetitively eluted with 50 mg/ml FLAG peptide (Sigma) in TBS/E (50 mMTris-Cl, pH 7.5, 150 mM NaCl, 1 mM EDTA) at room temperature. The elutewas concentrated and the buffer was replaced by PBS using 30 kD cut-offultrafiltration (Millipore). Finally the concentrated solution waspassed through 0.2 mm filter to remove self-aggregated polymers.

Characterization of Recombinant Laminin-521

Secreted laminin in medium and after purification was characterizedusing 3-8% gradient SDS-PAGE. Proteins were visualized using Syprostaining (Bio-Rad) or transferred onto PVDF (FIG. 3). The membranes wereprobed with antibodies described above. After washing, the membraneswere incubated with HRP-conjugated goat antibodies. The immunoreactivitywas detected by a chemiluminescent kit (Life Science Products) accordingto the manufacturer's instructions.

Methods

Human ES Cell Cultures.

Human ES cells were cultured on r-laminin-521-coated laboratory dishesin chemically defined O3 medium (described in Rodin et al., NatureBiotechnol., vol. 28, pp. 611-615 (2010)) at 37° C. in 5% CO2. Cellswere routinely passed once every 10-12 days by exposure to Trypsin-EDTAsolution (GIBCO Invitrogen) for 5 minutes at 37° C. They were thengently pipetted to break into single-cell suspension and defined trypsininhibitor (GIBCO Invitrogen) was added. The cell suspension wascentrifuged at for 4 minutes, the supernatant discarded, the cell pelletresuspended in prewarmed O3 medium, and cells were then passed through a40 μm sieve. After that cells were plated on new r-laminin-521-coateddishes at a concentration 30 Kcells/cm² (1:25-1:30 split ratio). Cellswere fed once a day with fresh medium prewarmed in an incubator for 1hour, except for the first day after a passage, when only a few drops offresh medium were added. Control cells of the same line were cultured onMatrigel (BD Biosciences) in O3 medium as described in Rodin et al.,Nature Biotechnol., vol. 28, pp. 611-615 (2010). Control cells werepassaged in pieces.

Cell Culture Dish Coating.

Ninety-six-well tissue cell culture plates were coated overnight at 4°C. with sterile solutions of the ECM proteins mouse LN-111 (Invitrogen),human recombinant LN-511, and human recombinant LN-521, all at aconcentration of 30 μg/ml (5 μg/cm²). For control cells, BD Matrigel™hESC qualified (BD Biosciences) was used according to the manufacturer'sinstructions.

Cell Adhesion Assay.

The assay was performed as described (Extracellular Matrix Protocols,2000). Briefly, 96-well plates were coated by extracellular matrixproteins as described above and blocked by O3 medium containing bovineserum albumin. The ES cells were plated at a cell density of 600cell/mm² upon extracellular matrix-coated plates and were left to adherefor either 1 hour or 1 day at the cell incubator. Non-adherent cellswere washed away, and adherent cells were fixed for 20 min by 5%glutaraldehyde, stained by 0.1% Crystal Violet.

Real-Time PCR Quantification of mRNAs.

Total RNA was isolated and cDNA was synthesized as described in Rodin etal., Nature Biotechnol., vol. 28, pp. 611-615 (2010). Real-timequantitative RT-PCR Taqman assays were performed using the AppliedBiosystems 7300 Real-Time PCR System. All reactions were done inquadruplicate with predeveloped gene expression assay mix (AppliedBiosystems) containing primers and a probe for the mRNA of interest.Additional reactions for each experiment included predeveloped geneexpression assay mix for GAPDH, used to normalize the RNA input. Alldata were analyzed with 7300 System SDS Software version 1.4.

FACS Analysis.

OCT4 expression was analyzed as described in Ludwig, T. E. et al.Derivation of human embryonic stem cells in defined conditions. Nat.Biotechnol. 24, 185-187 (2006). Cells were run on FACSCalibur FlowCytometer (Becton Dickinson). Data were analyzed with CellQuest software(Becton Dickinson).

Results for Example 1

To find out how different cell culture coatings affect single cellsurvival of human ES cells in O3 medium without any additives, wecompletely dissociated H5181 cells and plated them on either Matrigel,mouse laminin-111, human r-laminin-511, human r-laminin-521, or amixture of r-laminin-511 and r-laminin-521. The results are seen in FIG.4. As expected, almost no cells remained attached to Matrigel or mouselaminin-111 by 24 hours after plating. In contrast, the cells on humanr-laminin-521, the mixture, and at a less extent on human r-laminin-511survived.

The experiment was repeated on human ES cells in O3 medium treated withROCK inhibitor Y-27632. These results are seen in FIG. 5. The stem cellsremained attached on all 5 coatings.

To quantify this effect, we performed cell adhesion experiments on allof the above-mentioned coatings at 1 hour and 1 day after plating (i.e.,each of the five coatings, with and without Y-27632). FIG. 6 shows theresults for the 1-hour experiment for LN-521, LN-511, and Matrigel (MG)without Y-27632. The adhesion of human ES cells in O3 medium without anyadditives was roughly the same at this time point.

FIG. 7 shows the results for the 1-day experiment for all of thecoatings. The plates containing the Y-27632 inhibitor are labeled as“In&” along with the coating. At 1 day after plating, the stem cellsadhered to LN-521 without additives 20 times better than to Matrigelwithout additives. In addition, the results showed that the adhesion ofcells to LN-521 without additives was similar to the adhesion of cellson all dishes including the ROCK inhibitor. In other words, no ROCKinhibitor is necessary with LN-532 coating to obtain results similar tocoatings that do contain the ROCK inhibitor.

We cultured human ES cells on human r-laminin-521. passaging them aftercomplete dissociation into single cell suspension every 10-12 days in1:25 to 1:30 ratios. The cells proliferated robustly with a stable andhigh rate for at least 9 passages (3 months). Moreover, after 3 passages(1 month), they underwent the same number of cell doublings as stemcells after 20 passages (100 days) cultured using conventional methods.This is illustrated in FIG. 8, which shows the increase in the number ofcells versus time. The cells on LN-521 increased at a much faster ratethan on Matrigel. Thus, the new r-laminin-521 hES cell culture methodwas advantageous in terms of time and labor.

To confirm the identity of hES cells after several single cellsuspension passages on human r-laminin-521 in O3 medium without anyadditives, we performed FACS analysis for Oct4, a marker ofpluripotency. FIG. 9 shows the results for cells grown on r-LN-521,while FIG. 10 shows the results for cells grown on Matrigel. Thepercentage of positive cells is listed in parentheses. R-LN-521 had amuch higher percentage of pluripotent cells.

In addition, the number of mRNA transcripts of the pluripotency markersOct4 and Nanog was quantified. FIG. 11 shows these results. For bothmarkers, the cells on LN-521 showed higher numbers of transcripts.

Both methods showed that the new methods provides hES cells of the sameor better quality than the current method, which uses Matrigel as a cellculture dish coating material and passaging of the cells in smallclumps.

Example 2

In this Example 2, expressed and purified recombinant LN-521 that wasalso expressed in pluripotent hES cells was studied for its effects oncultured hES and iPS cells. The results showed that LN-521, alone,strongly supports self-renewal of pluripotent hES and iPS cells, and,importantly, it allows survival of pluripotent stem cells followingplating of trypsinized stem cells in single cell suspension on LN-521 athigh dilutions. The effects of LN-521 were shown to be mediated bysignaling via α6β1 integrin through induction of hES cell migration andPI3K/Akt pathway activation. The results may be applied for an effectiveand even automated expansion method for large-scale production ofpluripotent hES and iPS cells, as well as for development of new mediumformulations for self-renewal of pluripotent stem cells. The newhES/hiPS cell culture method described here closely resembles standardcell culture methods, e.g. that are used to culture fibroblasts, and,therefore, it does not demand special skills. It consumes significantlyless cell coating material per one cell division and is time efficient,thus providing significant economical profits in comparison with theprevious procedures for culturing hES/hiPS cells.

Human recombinant LN-521, which has previously not been available inpure form, was produced by cloning full-length laminin β2 cDNA andcarrying out a triple-transfection of HEK293 cells with the humanlaminin α5, β2 and γ1 chains. Human recombinant LN-111 and LN-121 weresimilarly generated following cloning of full-length α1 and β2 chaincDNAs and triple transfection of HEK293 cells with human α1, β1 and γ1and α1, β2 and γ1 chain cDNAs, respectively. The purified LN-521, LN-111and LN-121 proteins were shown to contain, respectively, pure α5, β2,and γ1 chains, α1, β1 and γ1 chains, as well as α1, β2 and γ1 chains, asshown by protein staining and Western blot analysis.

Methods

Human ES and iPS Cell Cultures.

Human ES cells of HS181 and HS401 were cultured on LN-521-coated culturedishes in O3 medium (described in Rodin et al., Nature Biotechnol., vol.28, pp. 611-615 (2010) with pH was adjusted to 7.35), mTeSR1 (STEMCELLTechnologies) and chemically defined and xeno-free TeSR2 (STEMCELLTechnologies) at 37° C., 5% CO₂. Initially, cells of the lines weretransferred onto a LN-521 coating from a human feeder cell layer bycareful scratching using a sterile knife with subsequent trypsinization,or the cells were trypsinized into single cell suspension from a LN-511coating. The cells were provided once a day with fresh medium pre-warmedin an incubator for one hour, except for the first day after plating,when only a few drops of fresh medium were added. Cells were routinelypassed once in 10-12 days by exposure to Trypsin/EDTA (GIBCO InvitrogenCorporation, Paisley, Scotland) for 5 minutes at 37° C., 5% CO₂. Theywere then gently pipetted to break into single-cell suspension and aDefined Trypsin Inhibitor (GIBCO Invitrogen) was added. The cellsuspension was centrifuged at 25 rcf for 4 minutes, the supernatant wasdiscarded, the cell pellet was resuspended in prewarmed O3 medium, andthe cells were then passed through a 40 μm sieve. Subsequently, thecells were plated on new LN-521-coated dishes at a concentration of30,000 cells/cm² in 1:25-1:30 split ratios. Control cells of the sameline were cultured on Matrigel (STEMCELL Technologies) and LN-511 in O3medium as described previously.⁹

For defined and xeno-free cultures, TeSR2 (STEMCELL Technologies) mediumand defined free from any animal derived component TrypLE™ Select (GIBCOInvitrogen Corporation, Paisley, Scotland) enzyme were used. The cellswere passed every 10-14 days by exposure to TrypLE™ Select for 4-5minutes at 37° C., 5% CO₂. Then, the enzyme was carefully aspirated andprewarmed TeSR2 medium was added. After that, the cells were gentlypipetted to break into single-cell suspension, centrifuged, thesupernatant was discarded, and the cell pellet was resuspended inprewarmed TeSR2. The cells were then passed through a 40 μm sieve andplated on a fresh LN-521 coated dish.

Tissue cell culture plates were coated overnight at 4° C. with sterilesolutions of the ECM proteins, such as human recombinant LN-521, all ata concentration of 30 μg/ml (5 μg/cm²). Control plates were coated withMatrigel according to the STEMCELL Technologies' instructions. Prior touse, dishes were pre-warmed in an incubator for one hour after whichprewarmed O3 medium was added without additional washing of the dish.After applying a fresh LN-521 solution to a new dish, the remainingLN-521 solution could be used at least once more. All the adhesion,survival, and inhibition of survival experiments were carried out usingfresh coating materials.

The human iPS cells, ChiPSW line, were derived from lentivirallytransduced human foreskin fibroblasts (HFF) with OCT-4/SOX2/NANOG/LIN28reprogramming genes. The ChiPSW line had normal male karyotype, 46,XY,in repeated testing at different passages. Prior to cell culture andpassaging experiments, the cells were first characterized in vitro forexpression of pluripotency markers. Immunofluorescence study withantibodies against Oct3/4 (SC-5279), Nanog (SC-33759), TRA-1-60(SC-21705) and SSEA4 (sc-21704) showed that the cells expressed allthose markers of pluripotency. RT-PCR analysis confirmed that the cellslacked expression of the viral transgenes. The pluripotency of ChiPSWcells was confirmed by in vitro (embryoid bodies formation andimmunofluorescence study) and in vivo (injection subcutaneously intoSCID beige mice) experiments. Cells of ChiSW line could be furtherdifferentiated into beating cardiomyocytes. They were also capable ofundergoing hematopoietic differentiation.

Before taken to the present feeder free cultures, the iPS cells weremaintained and expanded over irradiated human foreskin fibroblasts.Knock-out serum (KSR, Invitrogen)-supplemented media was used forpropagation, supplemented with 8 ng/ml of basic fibroblast growth factor(bFGF, R&D Systems). Cells were fed on a daily basis and weekly passagedusing collagenase IV (1 mg/ml, Roche) or manual dissection whenrequired.

Prior to these experiments, the CutB1.2 cells were shown to expresspluripotency markers Oct4/Nanog/Sox2/TRA-1-60/TRA-1-81, to lackexpression of the viral transgenes, and the pluripotency of the cellswas confirmed both by in vivo and in vitro differentiation studies.

Laminin-521 and Other Coating Materials.

Human recombinant LN-521, available from BioLamina, AB, Stockholm(www.biolamina.com), was produced in human embryonic kidney cells(HEK293; ATCC CRL-1573) sequentially transfected with full-lengthlaminin γ1, β2 and α5 constructs essentially as described previously.For protein production, the HEK293 cells were cultured in Dulbecco'smodified Eagle's medium (DMEM) supplemented with GlutaMax I for up tosix days. The LN-521 molecules were affinity-purified using anti-FLAGmatrix (Sigma), and then characterized using 3-8% and 4-15% gradientSDS-PAGE under reducing and nonreducing conditions. The proteins werevisualized using Sypro Ruby (Bio-Rad) protein staining andimmunostaining of the chains on polyvinylidene difluoride membranes. Tofurther characterize the protein, Western blot analysis with antibodiesagainst the laminin α5, β2 and γ1 chains was performed. Humanrecombinant laminin-111 and laminin-121 were produced similarly toLN-521 and shown to contain the correct chains of predicted molecularsizes by Western and SDS-PAGE. If not otherwise stated, thecorresponding mouse laminin-111 (Invitrogen) was indicated as LN-111.

Reagents and Antibodies.

InSolution™ LY 294002 (a specific Akt inhibitor), InSolution™ Wortmannin(a specific PI3K inhibitor), and InSolution™ 98059 (a specific MEK1/Erkinhibitor) were purchased from Calbiochem. Antibodies to phospho-Akt(#4060), total-Akt (#9272), phospho-Erk (#9101), and total-Erk (#9102)were obtained from Cell Signaling Technology. PathScan Phospho-Akt1sandwich ELISA kit (#7160), PathScan total Akt1 sandwich ELISA kit(#7170), PathScan Phospho-Akt2 sandwich ELISA kit (#7048), and PathScantotal Akt2 sandwich ELISA kit (#7046) were also purchased from CellSignaling Technology. Antibodies to Calnexin (#ab10286) were obtainedfrom Abcam. Function blocking antibodies to various integrin subunits,mouse isotype antibodies, and α-dystroglycan were purchased fromMillipore. Since function blocking antibodies to aV (MAB1980) and α6(MAB1378) were presented in a solution with sodium azide, before theinhibition of survival experiment all the antibodies to alpha integrinsubunits were dialyzed thrice against O3 medium for 2 hours each time.Antibodies to Lutheran receptor and α-fetoprotein, as well as ratisotype control antibodies were obtained from R&D Systems. Antibodies toOct4, Nanog, SSEA-4, smooth muscle actin and MAP-2 were purchased fromMillipore.

Immunofluorescence.

For immunofluorescence studies, ES cells were cultured and fixed in8-well slide chambers (BD Biosciences) or 96-well plate wells by 4%paraformaldehyde, permeabilized by 0.1% Triton-X and blocked by 10%bovine fetal serum (GIBCO Invitrogen Corporation) in phosphate-salinebuffer (PBS) containing 0.1% Tween-20 (Sigma-Aldrich, St. Louis,http://www.sigmaaldrich.com) for one hour. Incubation with primaryantibody was performed for 1.5 hours at room temperature. Incubationwith secondary antibody and 4,6-diamidino-2-phenylindole (DAPI,Molecular Probes) was performed for 40 minutes. Between incubations, thespecimens were washed with 0.1% Tween-20 in PBS buffer three to fivetimes. Specimens were preserved in a fluorescence mounting medium (Dako,Glostrup, Denmark, http://www.dako.com), and observed under afluorescence microscope (Leica, Heerbrugg, Switzerland,http://www.leica.com).

Real-Time PCR Quantification of Different mRNAs.

Total RNA was isolated using Absolutely RNA Microprep Kit (Stratagene,La Jolla Calif., www.stratagene.com) according to the manufacturer'sinstructions. cDNA was synthesized using 0.2 μg of total RNA in 20 μlreaction mixture, containing oligo(dT)12-18 primers and Superscript IIreverse transcriptase (GIBCO Invitrogen Corporation), according to themanufacturer's instructions. Real-time quantitative RT-PCR Taqman assayswere performed using the Applied Biosystems 7300 Real-Time PCR System(Applied Biosystems, Foster City, Calif.). All reactions were done inquadruplicates with the use of a pre-developed gene expression assay mix(Applied Biosystems) containing primers and a probe for the mRNA ofinterest. Additional reactions for each experiment includedpre-developed gene expression assay mix for GAPDH for normalizing theRNA input. All data was analyzed with 7300 System SDS Software v 1.4.

FACS Analysis.

Cells were removed from the culture dish with Trypsin/EDTA, dissociatedinto single cell suspension and resuspended in ice-cold FACS buffer (2%fetal bovine serum, 0.1% sodium azid in Hank's buffer). Incubation withprimary antibodies against SSEA-4 (from Millipore, Billerica, Mass.,http://www.millipore.com) was performed for one hour on ice. Then, thecells were washed three times with ice-cold FACS buffer. Subsequently,the cells were probed in FACS buffer with 1:400 dilution of Alexa Fluoranti-mouse secondary antibodies (GIBCO Invitrogen Corporation) for 30minutes in the dark, and washed four times. Control cells were incubatedwith mouse immunoglobulins and, subsequently, with the secondaryantibody as described above. Cells were analyzed on FACSCalibur FlowCytometer (Becton Dickinson, San Jose, Calif.). Data were analyzed withthe CellQuest software (Becton Dickinson).

Karyotyping.

Karyotyping of the cell lines was carried out using standard Q-bandingtechniques. Samples of cells were treated with colcemid KaryoMAX (0.1μg/ml; Gibco Invitrogen Corporation) for up to 4 hours, followed bydissociation with Trypsin/EDTA solution (Gibco Invitrogen Corporation).The cells were pelleted via centrifugation and re-suspended inpre-warmed 0.0375 M KCl hypotonic solution and incubated for 10 minutes.Following centrifugation, the cells were resuspended in fixative (3:1methanol:acetic acid). Metaphase spreads were prepared on glassmicroscope slides and G-banded by brief exposure to trypsin and stainedwith 4:1 Gurr's/Leishmann's stain (Sigma-Aldrich Co.). A minimum of 10metaphase spreads were analyzed and additional 20 were counted.

Teratoma Formation.

Teratoma formation experiments were performed by implantation ofapproximately 10⁶ cells beneath the testicular capsule of a young(7-week-old) severe combined immunodeficiency (SCID) mouse. Threeanimals per each cell line were used. Teratoma growth was observed byweekly palpation, and the mice were sacrificed eight weeks after theimplantation. The teratomas were fixed, and sections were stained withhematoxylin and eosin (HE) or with hematoxylin, eosin and PAS (HE-PAS).The presence of tissue components of all three embryonic germ linelayers was demonstrated, as analyzed from the stained sections. Allanimal experiments were performed at the infection-free animal facilityof the Karolinska University Hospital in accordance with ethicalcommittee approval.

Embryoid Body Formation.

ES cells from LN-521 coated cell culture dishes were exposed to TrypLE™Select for one minute at 37° C., 5% CO₂, washes two times with a medium,broken into large pieces and cultured in suspension in low adhesionplates. The medium used for this was Knockout DMEM (GIBCO InvitrogenCorporation) supplemented with 2 mM L-glutamine, 20% fetal calf serum(GIBCO Invitrogen Corporation), 0.1 mM β-mercaptoethanol (GIBCOInvitrogen Corporation) and 1% non-essential amino acids (GIBCOInvitrogen Corporation). After 1-2 weeks in suspension, the embryoidbodies were transferred onto gelatin coated tissue cell culture 96-wellplates (Sarstedt), cultured for 1-2 weeks, then fixed, stained withantibodies against markers of all three embryonic germ line layers(smooth muscle actin, MAP-2 and α-fetoprotein) and analyzed as describedabove for immunofluorescence.

Cell Adhesion Assay.

Briefly, 96-well plates were coated by extracellular matrix proteins asdescribed above and blocked by O3 medium containing bovine serumalbumin. The ES cells were plated at cell density of 50,000 cells/cm²onto extracellular matrix-coated plates and left to adhere for 1 hour ina cell incubator. After that the plates were washed 3 times with themedium to remove the non-adherent cells, and then the adherent cellswere fixed for 20 minutes by 5% glutaraldehyde, stained by 0.1% CrystalViolet. (Kebo Lab, Spanga, Sweden, http://www.kebolab.se). After onehour and 3 washes with water, Crystal Violet was extracted with 10%acetic acid and quantified by measuring optical density at 570 nm. Allthe experiments were performed in quadruplicate.

Cell Survival and Inhibition of Survival Assays.

The survival assay was performed as described for the cell adhesionassay above, except that the cells were left in the cell incubator for24 hours. For inhibition of survival assay, the cells were kept in amedium with function blocking antibodies at the concentrationsrecommended by the manufacturer or pathway inhibitors at concentrationsindicated in the text for 30 minutes, and then plated on the coateddishes. All the experiments were performed in quadruplicate.

Western Blotting and ELISA.

HS 181 cells were trypsinized into single cell suspension as describedabove. For inhibition experiments, the cells were kept in O3 medium withblocking antibodies or pathway inhibitors for 30 minutes and then 450Kcells were plated on 35 mm dishes precoated with the appropriate matrixcoating. For other experiments, same number of cells was plated directlyafter trypsinization. In all cases the cells were allowed to spread for1 hour at 37° C., 5% CO₂. After two washings in ice-cold PBS, the plateswith cells were snap frozen in liquid nitrogen and stored at −80° C. Toprepare samples for western blots and ELISA, the plates were slowlythawed and kept on ice with 100-150 μl of lysis buffer (50 mM Tris-HCl,pH7.5, 150 mM NaCl, 0.5% deoxycholate, 0.5% SDS, 1% Triton X-100, 1%Igepal, Complete™ (Roche) and Phospho-Stop™ (Roche)) on top. Then, thecells were scraped, pipetted and sheared through a 27G ¾″ needle. Afterthat, the cell pellets were clarified by centrifugation at 16,100 rcffor 15 minutes at 4° C. For western blots, 4-12% gradient gels were usedfor SDS electrophoresis and the proteins were transferred to PVDFmembranes. The membrane was hybridized with the antibody of interestaccording to the manufacturer's instructions. ChemoluminescentHRP-substrate from Amersham Biosciences was used for visualization. Forthe densitometry analysis the films were scanned at 2,400 dpi andanalyzed by the Chemilmager5500 program (1D-Multi Line densitometrymode). For ELISA the samples were applied to the wells according to themanufacturer's instructions.

In Vivo Imaging and Migration Assay.

24-well plates were coated by extracellular matrix proteins and blockedby O3 medium containing bovine serum albumin. The ES cells were platedat cell density of 30,000-40,000 cells/cm² onto extracellularmatrix-coated plates and left to adhere for half an hour in a cellincubator. After that, the plate was transferred into high contentimaging system Operetta (PerkinElmer) equipped with environmentalcontrol unit, which allowed to keep 37° C., 5% CO₂. For two movies thatwere made, the brightfield images were taken once in 15 minutes during24 hours after plating using Harmony software (PerkinElmer), exported,and analyzed using ImageJ software (NIH, the US). For migration assaythe images were taken every seven minutes during 18 hours after plating.Images acquired between the fifth and seventh hours after plating wereanalyzed using MTrackJ plug-in (University Medical Center, Rotterdam,The Netherlands). 100 attached cells on each coating were traced andmean distances from the current to the previous point of the track werecalculated. Error bars show standard error of the mean s.e.m. (n=100).

Statistics. Statistical significance was determined the by Student'stwo-tailed t-test for unequal variances.

Discussion of Results for Example 2

Pluripotent hES cells express α1, α5, β1, β2 and γ1 laminin chains. Tocompare the adhesion and clonal survival of hES cells plated from singlecell suspension to different coating substrata, hES cells growing asmonolayers on LN-511 or as clusters on a feeder layer were trypsinizedinto single cell suspension in O3 medium and plated on cell culturedishes coated with Matrigel, LN-111, LN-511, LN-521 or a mixture ofLN-511 and LN-521, in the absence or presence of a ROCK inhibitor(Y-27632) and analyzed after 24 hours.

The cells did not survive on Matrigel or LN-111, but they did so assingle cells on human recombinant LN-511 and LN-521, as well as on themixture of the two. However, the survival of cells on LN-521 wassignificantly higher than on LN-511 (compared in numbers below). Cellsplated on human recombinant LN-111 or LN-121 failed to survive after 24hours (data not shown), which demonstrates that the presence of the β2chain in a laminin trimer, as such, is not sufficient to support theeffect. In the presence of ROCK inhibitor Y-27632, the hES cellssurvived on all surfaces.

There were, however, clear differences with regard to cell shapesbetween cells growing on LN-521 in the absence or presence of ROCKinhibitor 24 hours after plating. In the absence of ROCK inhibitor 24hours after plating, cells growing on LN-521 were round, while the cellsgrowing in the presence of ROCK inhibitor adopted spindle or crescentlike shape possibly caused by rearrangement of the actin cytoskeleton.

To test if LN-521 could be used as a cell coating material for long-termself-renewal of human pluripotent cells, HS181, HS401, H1 cells andhuman iPS ChiPSW and CutB1.2 cells were cultured on the protein in O3,mTeSR1 or TeSR2 media. Cells growing in O3 or TeSR1 media were passed insingle cells suspension every 10-14 days at ratios of 1:20-1:30.Pluripotent hES cells proliferated at a stable rate similar or higher tothat of cells grown on LN-511 or Matrigel when passed in small clumps.Thus, one passage on LN-521 yielded the same or higher number of celldivisions than that of control cells passed twice in clumps. HS181,HS401, and H1 cells proliferated for at least 24, 5 and 15 passages,respectively (9, 2 and 6 months) in an O3 medium. CutB1.2 iPS and ChiPSWcells have been cultured for 5 and 3 passages in mTeSR1, respectively.Interestingly, dissociated hES cells could be cultured on LN-521 undercompletely defined and xeno-free conditions, using TeSR2 medium andTrypLE Select enzyme. The plating efficacy after a passage was slightlylower than that of the cells in O3 or mTeSR1 media, and the dissociatedcells were passed normally every 10-14 days in 1:15-1:20 ratios. H1 andHS401 cells have been cultured for 12 and 4 passages, respectively (5and 1.5 months) in TeSR2.

The hES cells were usually plated in single cell suspension at 30,000cells per 1 cm² of culture dish coated with LN-521, after whichindividual cells lacked any direct contacts with each other. Eight hoursafter plating, the hES cells could be observed as single cellsexpressing pluripotency markers Oct4, Nanog, and Sox2. Twenty four hoursafter plating, the cells formed small monolayer colonies that eventuallycombined into large islands of monolayers covering most of the well.

Cells grown on LN-521 showed stable expression levels of pluripotencymarkers Oct4, Nanog, and SSEA4, which were similar to those in cellsplated on Matrigel and passed as small clumps. To compare the level ofspontaneous differentiation in LN-521 and Matrigel cultures, the amountof mRNAs for differentiation markers PAX6, SOX17 and SOX7 expressed inMatrigel cultures and in LN-521 cultures were compared after one (10days) and 10 passages (4 months). Quantitative RT-PCR revealed similaror less levels of expression of all three markers of differentiation inLN-521 cultures independently on the passage number.

Karyotypes were confirmed to be normal for hES cell lines HS181 and H1after 12 and 10 passages, respectively, on LN-521 in the O3 medium; andfor H1 after 7 passages in TeSR2 medium. Histological examination ofteratomas formed in SCID mice after injection of HS181 and H1 cellscultured for 13 and 12 passages, respectively, on LN-521 in O3 medium,and H1 cells cultured for 7 passages on LN-521 in TeSR2 revealeddevelopment of tissues containing all three germ lineages of the humanembryo. Differentiation in vitro also revealed, that the cells in allthree cases retained the competence to form embryoid bodies expressingmarkers of mesoderm (smooth-muscle actin), ectoderm (MAP-2) and endoderm(α-fetoprotein).

To quantify the efficacy of the coating substrata, adhesion after onehour and survival after 24 hours of dissociated hES cells on Matrigel,LN-111, LN-511, LN-521, and an equal mixture of LN-511 and LN-521 werestudied. Interestingly, after one hour about the same 75-80% of cellshad adhered to all the coatings, although the spreading of the cells wasclearly better on LN-521 and LN-511 than on Matrigel or LN-111 (notshown). After 24 hours, almost no cells had survived on Matrigel orLN-111, and very few on LN-511. In contrast, the survival of hES cellson LN-521 was approximately 20 times higher than on Matrigel, and threetimes higher than on LN-511.

To qualitatively compare the effects of LN-521 and ROCK inhibitor(Y-27632), we plated cells from the same single cell suspension at thesame plating density (40,000 cells per 1 cm²) in a medium containing 10μM of Y-27632 and studied their survival on the different coatings. Theuntreated cells on LN-521 and Y-27632 treated cells on Matrigel showedsimilar survival rate 24 hours after plating. Interestingly, evenY-27632 treated human ES cells survived better on LN-521 than onMatrigel.

If exogenous bFGF was removed from the O3 medium, the cells still showed20 times higher survival on LN-521 than on Matrigel. Moreover, hES cellssurvived on LN-521 24 hours after plating, even in a medium lacking allthe growth factors of the TeSR1 formulation (bFGF, LiCl, γ-aminobutyricacid (GABA), pipecolic acid and TGFβ¹⁶, suggesting that the survivalmechanism is independent of signaling induced by those factors.

An assay for inhibition of cell survival using function-blockingantibodies to potential receptors for LN-521 on the plasma membraneshowed that antibodies to integrins α6, and to a slightly lesser extentto β1, inhibited survival of human ES cells on LN-521. Function-blockingantibodies to other tested integrin subunits, as well as to Lutheranreceptor and α-dystroglycan, showed very little if any effects on humanES cell survival on LN-521.

Recently, it has been shown that ROCK inhibitors and blebbistatin actthrough abrogation of actin-myosin contractility, which is mediated byphosphorylation of the myosin light chain (MLC). To test if LN-521 hadsimilar activity, levels of phosphorylated MLC were compared between thedissociated cells on Matrigel, LN-111, LN-511, and LN-521 one and sixhours after plating (FIG. 12 and FIG. 13). Interestingly, western blotshowed that phosphorylation of MLC was even higher in the cells onLN-511 and LN-521 than that in the cells on Matrigel and LN-111. Sinceactin-myosin rearrangements are essential not only for contractions, butalso for cell motility, we surmised that the cell migration could becaused by interaction between LN-521 and its integrin receptor α6β1. Invivo imaging of the cells on Matrigel and LN-521 revealed that the cellsmigrated much faster on the latter and survived by aggregation intosmall fast moving colonies. Migration of hES cells on the four coatingswas also compared between the fifth and seventh hours after plating whenthe cells were still attached in all cases (FIG. 14). Motility of thehES cells on LN-521 was higher than that of the cells on other coatingsand correlated with the ability to survive on them. Treatment withfunction blocking antibody to integrin β1 significantly reduced motility(FIG. 15) and adhesion (data not shown) of the cells on LN-521.

An extensive body of data has shown that activation of the MEK1/Erk orPI3-kinase/Akt pathways by integrins can block anoikis. The effects ofLY 294002 and PD 98059, specific inhibitors of Akt and MEK1,respectively, were examined to explore the potential role of thesepathways in hES cell survival on LN-521. Blockade of Akt activation byLY 294002 was found to facilitate detachment and hES cell death, with nocells surviving 24 hours after plating on LN-521 (FIG. 16). In contrast,treatment with PD 98059 did not affect the survival that severely. hEScells treated with another PI3K/Akt specific inhibitor, Wortmannin, alsofailed to survive 24 hours after plating on LN-521, confirming thatactivation of PI3K/Akt was necessary for the cell survival on LN-521.

Since bFGF is known to be a potent activator of the MEK1/Erk pathway,the influence of which cannot be fully inhibited by PD 98059, weperformed the same experiment in O3 medium without exogenous bFGF withthe same result (FIG. 17). The efficacy of LY 294002 and PD 98059treatment was confirmed by western blot analysis of the treated andcontrol cells collected one hour after plating on LN-521 (FIG. 18 andFIG. 19).

Western blot analysis of extracts of cells growing on Matrigel andLN-521 with antibodies to phospho-Akt showed that the PI3K/Akt pathwaywas active in both cases (data not shown). To determine the levels ofAkt activation in the cells on different coating, ELISA was performed oncell lysates collected one hour after plating on Matrigel, LN-111,LN-511 and LN-521 when the cells still did not have direct contacts witheach other (FIG. 20 and FIG. 21). The level of Akt2 phosphorylation inthe cells on different coatings correlated with survivability on them.

It has been demonstrated that α6 and β1 integrins are the mostabundantly expressed integrin isoforms in human ES cells among alpha andbeta subunits respectively. Integrin α6β1 shows a broad spectrum ofspecificity towards different laminins, but the binding affinity forLN-521 or LN-511 is higher than that for LN-111. Recently, it has beenestablished that (32 laminins have higher affinity for integrins thanthe β1 laminins. Therefore, having the highest affinity for theintegrin, LN-521 can provide the best anchorage for migration and canconvey the highest dose of signal via α6β1 integrin resulting in thebest survivability of dissociated pluripotent hES cells, although e.g.LN-511 also can do it at a lesser extent.

The results of this work may facilitate culturing and expansion ofpluripotent human stem cells in general, and even make automatedexpansion of such cells possible including cell aimed for clinicalapplications. The survival of dissociated hES cells on LN-521 appears tobe dependent on migration and the cells can therefore not survive afterplating at ultralow densities. The new hES cell culture method describedhere utilizes only a naturally occurring LN-521 adhesion protein thatdoes not damage the cytoskeleton as ROCK inhibitor or blebbistatintreatment do. Thus, LN-521 most probably favors survival of the cellsonly with the correct integrin profile on the cell surface. The presentresults also showed that hES cells plated on LN-521 at relatively lowdensities of 20,000-30,000 cells per 1 cm² could survive and multiply atleast as efficiently as in the other hES culture systems. The new methodclosely resembles standard cell culture procedures, e.g. culturing offibroblasts, and, therefore, hES cell cultures on LN-521 do not demandspecially trained personnel, which has been a major problem before sinceculturing of hES cells has been a technological challenge.

The widely used TeSR1 formulation for human pluripotent stem cellself-renewal was initially developed for use with Matrigel and itcontains high doses of bFGF that mostly targets the MEK1/Erk pathway,which is not widely considered to have a beneficial effect forpluripotent hES cells. The present results can lead to development ofnew medium formulations utilizing benefits of LN-521 as coating materialand specifically targeting pathways, which are important for human EScell self-renewal.

In summary, the present work has demonstrated that LN-521, normallysecreted by pluripotent hES, can as a sole coating material supportself-renewal of human pluripotent stem cells in culture, similar toLN-511. Both laminins facilitated growth of the cells as homogenousmonolayers in vitro. However, an important difference between the twolaminins is that hES/hiPS cells could be trypsinized into single cellsuspension, and plated and effectively expanded from single cells onLN-521, as opposed to manual splitting of cell clusters, as is currentlyrequired for expansion of hES/iPS cells grown on Matrigel or feedercells. The results of this work may facilitate culturing of pluripotenthuman stem cells and facilitate automated expansion of such cells.

Example 3

Human embryonic stem cells were cultured upon a laminin-521 substrate intwo different cell culture mediums. The two cell culture mediumsdiffered in the amount of bFGF, 3.9 ng/ml and 100 ng/ml.

FIG. 22 shows the growth curve of HS181 hES cells cultured in an O3medium comprising 3.9 ng/mL of bFGF with a laminin-521 substrate after 5passages (40 days) in solid black. The O3 medium was a variant of thecommercially available chemically defined mTeSR1 medium with bovineserum albumin as the only animal derived component. The growth curve ofHS181 hES cells cultured in an O3 medium comprising 100 ng/mL of bFGFwith a laminin-521 substrate is shown in dashes. The cells weredissociated into single cell suspension for passaging. As seen here, thegrowth curve for the lower amount of bFGF was as good as or better thanthe higher amount of bFGF.

FIG. 23 shows the relative amount of mRNA transcripts for pluripotencymarkers Oct4 and Nanog after 5 passages (40 days) for both media, whichwas obtained using real-time quantitative reverse transcriptionpolymerase chain reaction (RT-PCR) analysis. Again, similar amounts wereobtained in both cell culture mediums, indicating that the stem cells inthe lower amount of bFGF maintained pluripotency. Thus, a lower amountof bFGF can be used and still obtain good results, particularly incombination with the laminin-521 substrate.

Example 4

In Examples 1 and 2, the dissociated stem cells survived mostly throughactive motility on LN-521 and association into small effectively growingand migrating monolayer islands. The cells plated at very low, cloningdensities died through anoikis, a specialized from of programmed celldeath. Normally, integrin-related signaling from extracellular matrixmolecules, e.g. laminins, and cadherin-related cell-cell signaling, canprevent anoikis. The most abundant cadherin isoform on the human ES cellsurface is epithelial-cadherin (E-Cadherin). The experiments in Examples3 were performed to find out if a combination of LN-521 and E-Cadherincould protect human ES cells from anoikis and allow clonal survival ofthe cells.

hES cells were plated in mTeSR1 medium on different coatings at adensity of 250 cells per cm² and monitored after 5 days in culture usingan alkaline phosphatase staining kit. Neither laminin-521 nor E-Cadherinalone permitted efficient clonal survival of individualized hES cells.

Next, combinations of laminin-521 and E-Cadherin were tested todetermine whether the combinations could sustain clonal survival of thecells. Fixed amounts of laminin-521 were used for the cell culture dishcoating in combination with titrated E-Cadherin. Clonal survival ofcells was achieved at different ratios of E-Cadherin to laminin-521including from about 1:10 w/w to about 1:5 w/w.

Additional tests were performed to test the effects of variousmodifications to the mTeSR1 medium. A mixture of E-Cadherin tolaminin-521 in a ratio of about 1:10 w/w was used to coat the plates.Unexpectedly, it was discovered that a 2× increase in albuminconcentration significantly improved clonal survival of hES cells on thelaminin-521/E-Cadherin matrix. The rate of individualized hES cellsurvival under these conditions was from 10 to 15% and was at least oneorder of magnitude higher than that of the cells on Matrigel,laminin-521, or E-Cadherin alone in both mTeSR1 medium and mTeSR1 mediumwith additional albumin. Time-lapse photography of the cells confirmedthat laminin-521/E-Cadherin coatings facilitated cell survival throughproliferation of the single cells, not through the aggregation ofdifferent cells. Laminin-521/E-Cadherin similarly sustained clonalsurvival of cells in completely chemically defined and xeno-free TeSR2medium with the addition of recombinant human serum albumin (rHSA).

In additional testing, hES cell lines were derived. Twenty-four welltissue cell culture dish plates were washed twice with Dulbecco'sPhosphate Buffered Saline. Next, the dish plates were coated for 2 hoursat 37° C. with sterile solution containing: 48 μL of 100 μg/mLlaminin-521 (BioLamina AB, Stockholm); 6 μL of 82 μg/mL E-Cadherin(R@DSystems); and 300 μL of DPBS with calcium and magnesium (GIBCO) toproduce a laminin-521/E-Cadherin matrix.

The donated embryos used to derive the hES cell lines were obtained froman accredited in vitro fertilization clinic after the consent of bothpartners and ethics approval were obtained. Only fresh or frozenembryos, which could not be used for infertility treatment, were used inthe derivation procedures. One or two cells were isolated from an 8-cellstage embryo using a micropipette after making a small opening to thezona pellucida with a laser apparatus designed for this purpose. Thisprocedure has been accredited for clinical use in pre-implantationgenetic diagnostics (PGD). The cells were placed on thelaminin-521/E-Cadherin matrix in TeSR2 medium with additional rHSA. Thecells successfully attached and started to proliferate. The parentalembryos were allowed to grow to a blastocyst stage and frozen. Theembryo culture was carried out using xeno-free standardized in vitrofertilization culture media in drops under oil at 37° C. and 5% O₂/10%CO₂. After removal of the zona pellucida, the inner cell masses of fivehuman blastocysts were mechanically isolated and plated in 4-wellculture plates (Nunc) onto the laminin-521/E-Cadherin matrix. Followingan initial 48 hours of culture, the culture medium was replaced on adaily basis. After 10 to 14 days, the outgrowths were mechanicallyisolated and replated onto laminin-521/E-Cadherin matrix. Mechanicalpassaging was used for the subsequent 2 to 3 passages after whichcolonies were passaged using TrypLE Select (GIBCO).

Using the laminin-521/E-Cadherin matrix described above with the mTeSR1medium including additional bovine albumin, three new hES cell lineswere derived from six cultured blastocysts. Four days after plating, theinner cell masses gave stem cell-like outgrowths. FIG. 24 shows an earlystage derivation of a new human embryonic stem HS841 cell line onlaminin-521/E-Cadherin matrix. Morphologically typical human embryonicstem cells growing out from the inner cell mass of a day six blastocystfour days after mechanical isolation of the inner cell mass and platingon laminin-521/E-Cadherin matrix are shown.

Unexpectedly, the cell culture system and method gave stable, hES celllines in 3 out of 6 embryos (50%), a derivation rate higher than that ofstandard methods.

The use of a laminin-521/E-Cadherin matrix and TeSR2 medium withadditional rHSA, a completely chemically defined and xeno-freeenvironment, yielded similar results in the derivation of new hES celllines.

It will be appreciated that variants of the above-disclosed and otherfeatures and functions, or alternatives thereof, may be combined intomany other different systems or applications. Various presentlyunforeseen or unanticipated alternatives, modifications, variations orimprovements therein may be subsequently made by those skilled in theart which are also intended to be encompassed by the following claims.

1. A system for maintaining pluripotent stem cells, comprising: a cellculture medium comprising from greater than zero to 3.9 ng/mL of basicfibroblast growth factor (bFGF); and a substrate for providing supportto the stem cells.
 2. The system of claim 1, wherein the substratecontains laminin-521 or laminin-511.
 3. The system of claim 2, whereinthe substrate also contains a cadherin.
 4. The system of claim 1,wherein the substrate comprises laminin-521 and wherein the amount ofbFGF is 3.5 ng/mL or less.
 5. The system of claim 1, wherein the cellculture medium comprises from 0.5 to 3.5 ng/mL of bFGF.
 6. The system ofclaim 1, wherein the cell culture medium further comprises at least oneadditional growth factor, at least one trace mineral, and at least onelipid.
 7. A cell culture medium that provides nutrition to pluripotentstem cells, comprising from greater than zero to 3.9 ng/mL of basicfibroblast growth factor (bFGF).
 8. The cell culture medium of claim 7,comprising 3.5 ng/mL or less of bFGF.
 9. The cell culture medium ofclaim 7, comprising from 0.5 to 3.5 ng/mL of bFGF.
 10. The cell culturemedium of claim 7, further comprising at least one inorganic salt. 11.The cell culture medium of claim 7, further comprising at least onetrace mineral.
 12. The cell culture medium of claim 7, furthercomprising at least one energy substrate.
 13. The cell culture medium ofclaim 7, further comprising at least one lipid.
 14. The cell culturemedium of claim 7, further comprising at least one amino acid.
 15. Thecell culture medium of claim 7, further comprising at least one vitamin.16. The cell culture medium of claim 7, further comprising at least oneadditional growth factor.
 17. The cell culture medium of claim 7,further comprising at least one inorganic salt, at least one tracemineral, at least one energy substrate, at least one lipid, at least oneamino acid, and at least one vitamin.
 18. The cell culture medium ofclaim 7, wherein the cell culture medium does not contain any one of (1)albumin, (2) insulin or an insulin substitute, or (3) transferrin or atransferrin substitute.
 19. The cell culture medium of claim 7, furthercomprising albumin, insulin, lithium chloride, GABA, TGF beta 1,pipecolic acid, L-glutamine, MEM non-essential amino acid solution, andDMEM/F12 solution.
 20. The cell culture medium of claim 7, furthercomprising at least one additional growth factor, at least one tracemineral, and at least one lipid.